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Launchpad User Guide - Flow Cytometry Core Facility

1. For Help press F1 New Annotation Cut Copy Paste I t I Annotation Properties See inser Delete Annotate Text J3 Annotation Properties p Data Properties Tags bse oo Trace z Peak labelling Display Properties Font Arial 7 Bold Italics E Size 30 10 Underline I ag trl 0 H degress Colour Set Add peak Clear Ctrl U ae me E X lt Delete peak Clear all BEEN E gt amp Delete peaks Peaks toolbar eee Figure 5 21 Main window menu Graphical display sub windows Chapter 5 Window and menu guides Display contents windows E Spectrum Contents A gt Chromatogram Contents Dataset Trace Sample G7 T angio memnsO00ghem z fr a Smoothing Segments C Levet 4 1 i aie Front Intensity Profle Mass Average Largest View Stack Overlay set 15 640 Display multiple samples 56 Items in blue have no display contents Spectrum Distribution Calibration Reference Calibrant List Notes Summary Sequence Calculator Results Sequence Peak Match Instrument Record Information Auto Experiment Results Peptide Mass Fingerprint Results Mascot Search Results Intensity Mapping LC MALDI Quantitation Results Chromatogram lt ____ Distribution Contents Formula Scale Maca osonenigore a aati Pi Gam tos
2. Figure 42 33 Example of a peak match report Performing a peak match with database sequences Introduction To search through a database for possible matches open the Load Sequence window select the database to search and from the list select the database entries to compare Pressing Match on the Match Peaks window will sequentially load each database entry carry out the fragmentation and perform peak matching on the results Matched peaks are listed in the Peak Match window with the number of matched peaks and the sequence in which the matches were found By setting Search to All database entries all of the sequences in the selected database will be searched this is of course only really practical for smaller databases containing a low number of entries Chapter 42 Sequence Calculator Sequence Calculator references 1 Browne C A Bennett H P J and Solomon S The Isolation of Peptides by High Performance Liquid Chromatography Using Predicted Elution Positions Anal Biochem Vol 124 Pp 201 208 1982 Bull H B and Breese K Surface Tension of Amino Acid Solutions A Hydrophobicity Scale of the Amino Acid Residues Arch Biochem Biophys Vol 161 Pp 665 670 1974 Engelman D Steitz T and Goldman A Identifying Nonpolar Transbilayer Helices in Amino Acid Sequences of Membrane Proteins Ann Rev Biophys Chem Vol 15 Pp 321 353 1986 Hopp T P and Woods K R
3. Copy comments from gt fit bill angio 2 with theo z Clear comments Load file Save file Apply to Current dataset x Select Comments File Ix Look in 9 Comments z 0a fe 3 angio comm LastCommentsUsed comm My Recent Documents Cc Select Comments File 21x Desktop Seven Comments J BH 3 angio comm fe LastCommentsUsed comm My Documents My Recent Documents E Desktop File name My Documents Files of type Comment files comm er My Computer er My Computer Fone f E Save astype Comment files comm g Cmca Figure 5 4 Comments window File menu 39 Chapter 5 Window and menu guides View menu This menu controls the window which displays collected data and instrument status Instrument status is described in Checking the instrument status on page 91 A Spectrum Contents Display Options BE Dataset 1 angl1__0001 2 psd_angl1_0001 General Graphs Graph Text Text Report Cursors Peak Labels Auto generated labels Labet EINE Traces Profle Average J Process Peaks Label type Mass x View Stack Overlay Se 15 610 Display multiple samples Precision 2 Decimal Added label Resolutior g Precision 2 Decimal Angle 0 Sj degrees Size 30 a 10 Underlined r r r Window var
4. Figure 42 18 Colour Editor button for sequence panels Pressing the Colours button will display the Colour Editor window for Sequence Panels Figure 42 19 Select the colours required for the panels and press Apply To save the colour scheme for re use at a later date press the Save button 618 Introduction Chapter 42 Sequence Calculator colour Editor Sequence Panels Bee 1 Sequence panel a a 9 10 B Colour sets Load Load defaults Apply Figure 42 19 Colour Editor window for Sequence panels To empty a viewing panel and delete the sequence contained within it select Delete sequence from the Edit menu To clear the insertion markers within a viewing panel select Clear markers Saving sequences The Save option on the File menu is used to save the sequence in the selected viewing panel to a database file Figure 42 20 If the sequence in the selected viewing panel is linked to a sequence in another viewing panel then that sequence will also be included in the database entry for the selected sequence In short any Introduction 619 Chapter 42 Sequence Calculator sequences linked to the sequence being saved will be saved along with that sequence When the sequence is reloaded new panels will be created as needed for the linked sequences Sequence Calculator OL x File Edit View Settings Load Save Sequence Import My Recent Documents Desktop lt My Documents
5. E Apply to Samples lo ole Aneel seer Ree ol es Figure 5 13 Processing windows a Processing menu Chapter 5 Window and menu guides 2 Polymer Analysis BBE Tolerance soo 4 mDa gt Distributions Copolymers Theoretical resolution 10000 Use this value I Start group End group Monomer w Analyse Optional features and instrument specific items are in red Calibration Annotation Sequence Calculator Sequence Calculator of x File Edit View Settings Synchronist Name Untitled gt PPI Snc e RES O A N terminus Hydrogen H Monat fo C tetminus Hydroxy H 0 Fragmentation Singly charged m Generate Peak Markers Calculate Cation Pton H Digest Length 0 af Reference Editor Reference file selection Filename TOF2_mix Reference Editor E Dataset 1 Select mass 22 Angiotensin 2 Angiotensin 1 Gu 1 fib N acetyl renin ACTH_1 17 ACTH_18 39 ACTH_7 38 Edit reference Mass fo46 5422 Fomula Angiotensin2 Compounds Update Mass Resolution Calculate Monaisotopic z 6000 Insert Delete Clear Select Peaks Hi Dataset 1 angiotensinagen_digest_005 Delete masses Delete gt Selected h r Automatic z Masses oro tL Peso 31
6. If the equipment is contaminated label the pallet or box in accordance with laws covering the transport of dangerous substances 10 Give a copy of the declaration to the carrier You must tell the carrier if the equipment is contaminated 11 Seal the original declaration in a suitable envelope and attach the envelope securely to the outside of the equipment package WRITE YOUR RETURN AUTHORISATION NUMBER CLEARLY ON THE OUTSIDE OF THE ENVELOPE OR ON THE OUTSIDE OF THE EQUIPMENT PACKAGE Equipment return repair declaration Sample records Print out and use the following two pages KKRATOS A SHIMADZU GROUP COMPANY FAX BACK 44 0 161 888 4401 EQUIPMENT RETURN REPAIR DECLARATION You are required to Return Authorisation Number Know and Declare all the substances that have been used in this equipment Read the procedure DOC 102 before you complete this form When parts are being returned obtain a returns authorisation number and send or fax this form completed to your local Service Centre before the parts are sent SECTION 1 EQUIPMENT Equipment model and type number Serial Number Has the equipment been used tested or operated yes Go to Section 2 no Go to section 4 SECTION 2 SUBSTANCES USED IN THE EQUIPMENT Are any of the substances which have been used in this equipment Your Service Engineer Centre will not be able to carry out any work on your equipment if it is contaminated wi
7. Mass 252 24 Closest peak M dM 104 75 Figure 20 16 Optional cursors on the display Cursors 357 358 Chapter 20 Managing Data Displays Tolerance band cursors Cursors With Type set to Tolerance additional cursors can be set to show a tolerance window centred about the last moved cursor Enter the required tolerance window and tolerance units The units available are Table 20 4 Tolerance band cursor units ppm Parts per million ppt Parts per thousand mDa milli Daltons 1 1000 Dalton Da atomic mass units Daltons Figure 20 17 shows an example of tolerance band cursors These are useful with peak searching options which search for peaks within a given tolerance window around the point marked by the cursor and in calibration Chapter 20 Managing Data Displays Display Options BEI General Graphs Graph Text Text Report Cursors Peak Labels Type l Tolerance Tolerance o7 4 Da Mass upto F 3 SHarge up E 4 Width None Normal Display Window Height Graph Display window Join with Off Line ion Gate Colours I All displays J p14r_msms0003 MALDI MS OL x File Edit view Instrument Automation Processing Help bel AA 2 al x 4 Display Spectrum v Profiles 1 Masses 230 1078 e gt Data p14r_msms0003 F1 c 4 Mar 2005 10 06 Cal tof 4 Mar 2005 10 10 CID of 1533 78 Shimadzu Biotech Kompact MALDI 6 Y2 7 0 Beta 1 Mode reflectron_ms_ms Po
8. Method Editor eoocecene oO UJ 194 Chapter 15 Automated operation Print The Method Editor Print window provides functionality for automatically printing the Spectra Mass List currently displayed Window and or Sequence Report during automated acquisition To access the Print window select the Print label from the tree as shown in Figure 15 26 below Method Editor default mtd 1 x Sample Method Sample Method default mtd Parent Printing Vy Parent 2 Mg Acquisition Print Options a Raster VS Auto Quality M 3e Laser Firing Print After Average bd WIE Processing Text Scaling 100 v oe vi RR bs Calibration Printing Type Colour hd Peak Cleanup Monoisotopic Peak Picker Peak Fiteing Pin o Do Not Mascot Spectra El EY Applications Mass List 4 MQ Output 3 Gil wW IF EI MS MS Compare Sequence Sequence Report Oligo Analysis Window Export Report lon Finder EY Acquisition Properties a mE Peak Selection Paster Margins Laser Firing DY Processing Left jo Top 0 ree Obie Bs oD BURL Peak Cleanup Monoisotopic Peak Picker Bottom jo Peak Filtering Mascot MQ Output E Data Storage il Maii Save Method Load Method Method Editor Figure 15 26 Method Editor Print Items to be printed are displayed in the Print list items to be hidden from printing are displayed in t
9. Checking the instrument status 92 Chapter 11 Checking the instrument status Introduction Before any type of data collection can begin the instrument status should be checked to see whether the instrument is ready for data collection If an error occurs a warning message will appear on the base window status bar and also in the log window The instrument status can be checked by means of the Status window This is available from the View menu on the base window Select Instrument Status from the View menu Figure 11 1 J p14r_msmso0004 MALDI MS olx File Edit view Instrument Automation Processing Help Display Contents Options Camera Instrument Status Database Viewer Tile Manager Event Filters Introduction z v Toolbar Display Spectrum X Profiles 1 X Masses 12 1040 1 gt amen VY Status Bar v Display Toolbar v Basic Parameters Figure 11 1 View menu The Status window will appear This window displays the main elements of the instrument in a schematic diagram The diagram shows the valve positions and valve states electrical power supply units turbo pumps and the laser unit The colour of the individual units indicates their current state Blue indicates that the unit is off yellow that the unit is in a transient state such as the turbo pump accelerating up to speed Green indicates that the unit is ready and red indicates failure of a unit for whate
10. Le 1081 25 11 7 58 12 656 46 Mail Alte yal aa TAA Yi ha 700 800 900 1000 1100 1200 1300 Mass Charge For Help press F1 Compound Database Yiewer 1 x Dataset 1 p14 _msms0004 Category amine acid x Annotate mass V Report Masses as Average 7 Decimals None e4 Cursor mass Glycine Gly G lanine la Serine Ser S Valina Yanl U Cursor mass range Proline Pro P TA Valine Val Annotate Threonine Thr T Cysteine Cys C Isoleucine Ile I Leucine Leu L 405 406 Chapter 21 Compound Database Viewer Profiles fi X Masses 649 1 395 l gt I File Edit View Instrument Automation Processing Help bel amp Se 2 8 x x Display Spectrum Data p14r_msms0004 4 Mar 2005 13 47 Cal tof 4 Mar 2005 10 10 CID Shimadzu Biotech Kompact MALDI 6 V2 7 0 Beta 1 Mode reflectron_ms_ms Power 74 Gate 1520 00 1545 00 P Ext 1534 lnt 6 0 m sum 1325 m Profiles 25 245 Smooth Av 1 1357 18 100 804 775 56 97 872 02 968 99 105 98 1163 18 1259 85 158 4 747 56 663 67 854 9 886 24 11 7 58 1372 5 A88 983 86 1081 25 1274 74 Af Bd glog y d pHs 85 04 1184431 ik aa aah PAA In Vhon 800 900 1000 1100 1200 Mass Charge eselde 752 Mw 700 1300 For Help press F1 Figure 21 2 Annotation using the Database Viewer Chapter 22 Manual peak labelling Chapter 22 Manual pe
11. cceeeeeeeeee teeta eens 81 Putting comments with collected data scsccseseeeeeeeeeeeeeeeneeees 83 Kalakololireidi oln mE A T TT 84 Checking the instrument statuS ssssssssssnunnnnunnnnnnunnnnnnnnnnnnnn 91 Intro d CtiOM reinen eaa aA A a AA R 92 Axima Assurance instrument StatuS essseseereesrerrrerer rrene 94 Axima Confidence instrument StatuS s sssssesressrrerrerrrrrrreen 97 Axima Performance instrument status ssseeesseeererrrrerre 100 Axima Resonance instrument statuS sssssssssrrssrrerrrrrrrresrs 103 Introduction to displaying data sssssssnsnsnnnnnnnnnnnnnnnnnnnnnnnnnnn 107 lNErOdUCEION serinin a a a e 108 Displaying SpectrUmM ssssssssssssressrreressrrrrsrsrerresrrrnrnerennnreren 109 Multiple sample datasetsS c cece cee eee eee ee tetas teeta teen eed 112 Preparation for data collection ssssssssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 117 MtrOdUCHION si ovedeceec suey ec eteae exc acces neke ue ede deenea eoees hone enemies 118 Sample plates and plt files cece eee eect eee eee ees 121 Raster laser firind cceceee ee eee eee eee eee eee eet ee eee eee ates 132 Tuning for an ACQUISITION cece eee eee eee eee eee teeta 135 Collecting data from a sample sssssssssnnnnnnunnnnnnnnnnnnnnnnnnnnnnnnn 139 LMEPOGAUCHION ica eseed cccgtintee seth eis neue hee a e 140 Sample selection ccceee cece eect ee eee e teens eee teeta ee neea
12. vine e t u W E B D ot Le ls 82 e Chapter 20 Managing Data Displays Table 20 2 Display toolbar functions for graphical displays Normal action Shift Ctrl SS Ctrl Zoom the selected Zoom next display to the full display in the window size sequence Zoom next display in the sequence Unzoom return a zoomed display to normal size Scroll graph 10 to Show high end higher end of graph Scroll graph 10 to Show low end lower end of graph Show full graph range Double the 5 x the 10 x the currently currently currently displayed displayed displayed range range range Scroll profiles displayed Show last increasing by 10 of profiles in data currently displayed range Scroll profiles displayed Show first decreasing by 10 of profiles in data currently displayed range Show all profiles in data Double the 5 x the 10 x the current profile current current range profile profile range range Switch the cursors off in Switch off the selected display additional cursors Return the region Cancel all between the cursors to amplification normal scale Amplify the region Amplify x 20 Amplify x Amplify x between the cursors x 2 200 2000 The Display Toolbar 339 340 Chapter 20 Managing Data Displays P le eit ib e ie le Ca a Table 20 2 Display toolbar functions for graphical displays Normal action Amplify the region between the cursors x 5 Amplify the reg
13. Background Paste Paste Insert gt Insert Delete Delete gt Annotate Annotate Annotation erop ir Propertie Tags gt Tag Ctrl T Tags gt Peak labeling pee ens Add peak SSS lear n X Delete peak Delete peaks Peaks toolbar Figure 20 1 Display menus Table 20 1 Display menu options Menu Purpose Insert Adds new displays either as a Column to the right or as a Row below the currently selected display or inserts an Inset Delete Removes a Column or Row of displays of which the selected display is a member or removes an Inset display Annotate Shows the New Annotation window allowing annotation to be added to graphical displays Introduction 335 336 Chapter 20 Managing Data Displays Table 20 1 Display menu options Menu Purpose Annotation Shows the Annotation Properties window for Properties a multiple selection of custom labels Tags Adds or removes tags to peaks between a pair of cursors or clears all tags without the need for cursors Introduction Chapter 20 Managing Data Displays The Display Toolbar On the right hand side of the base window is a vertical panel bar called the Display toolbar It contains small icon buttons used in conjunction with the mouse and keyboard to provide the most commonly used display controls in one location It is always visible regardless of the type of display in the window To use the display toolbar bu
14. Camera Instrument Status Database Viewer Tile Manager Events Logged sin Fle Action View Help e Ame Ey fa Event Viewer Local Application 2 127 event s i 1 Date Sow PAN information 12 07 2007 MALD T als Bele information 12 07 2007 MaD information 12 07 2007 MALD G information 12 07 2007 MALD information 12 07 2007 MALD information 12 07 2007 MALD G information 12 07 2007 MALD information 12 07 2007 MALD Qeror 12 07 2007 Usere G information 12 07 2007 MALD information 12 07 2007 MALD 4 Figure 5 6 E Dataset 1 lt untited gt Category Amino acid hd Report Masses as Average X Annotate mass V Decimals None hd Glycine G Gly GC Alanine Ala A Uslinn U1 U Cursor mass range Threonine Thr T Tile Manager x Current Tile Layout Cea Insert Row Column Inset Display Settings Delete Row Column Inset All Insets View menu b View menu 4l 42 Chapter 5 Window and menu guides Instrument menu This menu provides access to the windows which govern the type of slide being used and instrument operating mode It also allows access to the pumping and venting controls and to the windows which collect data from the samples and govern how and when data is stored See Preparation for data collection on page 117 for details Acquisition L i Firing Exp Tech Auto Qualit
15. Chromatographic peak detection Peaks detected Order Update Descending Ascending Profiles Mass Both Profile tagai Height Mass Profiles i Apex mV Profiles Masses 101 250 11250 11549 2 751 1000 11100 5 1301 1450 11100 11549 7 1651 1900 11100 11699 35 Peak display Previous First Last Clear 4 peak s selected olnt 100 0 1 m mass 5401 to 6000 Smoothed 1 100 50 800 850 900 Profile Chapter 30 Chromatography Click left mouse button on a detected peak in the list and the selected display will be updated with a new profile range mass or both 950 1000 Figure 30 5 Displaying detected chromatographic peaks Spectrum displays can be used to view detected peaks in exactly the same way as chromatogram displays Set the base window Displays option to Spectrum and select the detected peaks as above the spectra will update automatically as each detected peak region is selected Chromatographic peak detection 509 Chapter 30 Chromatography Manual peak detection using cursors Peaks can be marked manually using the cursors on a chromatogram trace set the Method option to Cursor andona chromatogram display of collected data mark the edges of the peaks to be entered into the peak list using the range cursors Figure 30 6 J mar100004 MALDI MS oj x File Edit Yiew Instrument Automation Processing Help bel PIE 2 a x x Disp
16. lonising Radiation Generator This instrument is covered by lonising Radiation Regulations 1985 Laser radiation Moving the The Kratos Axima instruments are Class 1 laser products containing a Class 3b invisible laser The instrument must be used in accordance with the instructions in this set of manuals If the product is used in any other way than that prescribed then there will be a risk to health due to exposure to the direct or reflected invisible laser radiation The laser unit within the Axima instrument operates at a wavelength of 337nm and has a maximum average output of 6mW This invisible spectrum radiation can be dangerous and personnel should avoid direct exposure to the beam Any adjustments made beyond those indicated in these manuals may result in hazardous radiation exposure The maximum emission from the instrument with the outer covers removed or open and all other safety covers in place is 0 03 of the peak laser power There is no user access to the laser unit nor are there any laser adjustment controls The instrument must not be operated with the outer covers removed or open instrument Before moving the instrument switch off isolate from the mains and ensure that the turbomolecular pumps have completely stopped This takes 30 minutes if the instrument has not been vented Further check that the feet have been retracted When moving the instrument remember that it is heavy and ensure that adequate person
17. pli aey yaey SBIEUIESER 6 2085 6 EELS eyep paiey i4 paljdde siaiy ou y m ejeq 2 8 R 8 8 YF 8B AR 8 Do ing Figure 16 13 Peak data before and after apply Figure 16 12 filters Filtering specified peaks 262 Chapter 16 Cleaning up data The peak list in the Filters dialogue can be edited using the Append button to add new entries Existing entries can be removed using the Delete button or the entire list can be removed using the Clear button The Save and Load buttons are available to allow Filters files to be created loaded and saved The tolerance used to define the window around a filter mass can be specified in either Daltons or parts per million The Convert formulae as option can be used to set whether filters entered as chemical formulae are converted to average most abundant or monoisotopic masses There may be situations when peaks defined for filtering are still required when calibrating e g in a PMF experiment during AutoRun Peaks that have the Cal Flag box ticked are filtered in such a way For example the filter applied above will not filter out the 5720 6 peak when calibrating In addition Filter files can be created using a spread sheet word processor or other text editing program The algorithm initially finds any lt less than or gt greater than filters Should more than one set of these be encountered then the biggest lt filter and the smallest gt filte
18. wadrala a Search gt fan Quedraley Enzymes r earcl IT RoboHelp Office tence 2 g 7 Help and Support SmartDraw Log Window IT Soundmax P mams a amp s beg E Run Startup Pol a Polymers fm Symantec Client Security gt Sample Plate Editor Q Log Off bill volo View Express og fas F g Search o Turn Off Computer Introduction The archiver is a utility which makes the task of archiving and restoring data to and from the computer system as simple as possible It is designed to display the data files and other files in your system as graphical icons To start the Archiver select Archiver from the programs menu on the Taskbar Figure 32 1 Launchpad Click the Archive icon BE E Show on startup V Help B Search Archiver Or use the Start menu system Stunts Gurme Pemer fan Documentation gt gt I Accessories f Administrative Tools 2 Compounds Database 2 Configure Environment Element y Figure 32 1 Starting the Archiver The Archiver window has a folder tree which can be expanded or contracted in the same manner as that of the Windows Explorer file manager window J ust like the Windows Explorer the Archiver can be used to view the entire network The initial state Chapter 32 Archiving data is to display a partially expanded view of the drive containing the Home folder as defined in the Configuration Editor For most users who a
19. 314 Chapter 18 Protein peptide analysis using Mascot search engine 2 When the search is completed the results are displayed in your web browser for example Internet Explorer baec Mascot Search Results User Your name Email your nameGkratos co uk Search title Arabidopsis thaliana MS MS experiment mz 1722 MS data file Automatically uploaded data Database NCBInr 20060216 3292813 sequences 1128164434 residues Taxonomy Arabidopsis thaliana thale cress 53253 sequences Timestamp 1 Mar 2006 at 14 57 34 GMT Significant hits giJi14532616 putative aldose 1 epimerase Arabidopsis thaliana Probability Based Mowse Score Tons score is 10 Log P where P is the probability that the observed match is a random event Individual ions scores gt 27 indicate peptides with significant homology Individual ions scores gt 33 indicate identity or extensive homology p lt 0 05 Protein scores are derived from ions scores as a non probabilistic basis for ranking protein hits 5 WV a nat x 4 E p 2 amp 3 Z 60 80 100 Probability Based Mowse Score Peptide Summary Report Format As Peptide Summary H Help Significance threshold p lt bo Max number of hits 20 Standard scoring MudPIT scoring Ions score cut off fo Show sub sets Show pop ups Suppress pop ups Sort unassigned Decreasing Score Require bold red I Mascot MS MS searches Chapter 18 Protein peptide a
20. Mascot Search Investigation Data File C Program Files Shimadzu Biotech Launchpad Data Cu r C Program Files Shimadzu Biotech Launchpad Data Customers University of St Andrews 417 4E0023_0001 H18 1 C Program Files Shimadzu Biotech Launchpad Data Customers University of St Andrews 417 4E0023_0001 H18 C Program Files Shimadzu Biotech Launchpad Data Customers Uniyersity of St Andrews 417 4E0023_0001 H18 1 C Program Files Shimadzu Biotech Launchpad Data Customers Univedgity of St Andrews 417 4E0023_0001 H18 ers University of St Andrews 417 4E0023_0001 H18 S For Help press F1 Intensity Significance 100 230 1492 103 149553 491 102 157 2672 289 272999 028 Double click within the node 112 253 2297 301 257878 928 to display the mass list 120 196 11203 467 1346611 886 158 196 1767 667 279637 846 173 220 2022 175 350281 164 175 272 5979 452 1048030 556 201 217 5161 040 1038488 969 229 186 3811 186 873470 500 230 220 2387 799 549719 105 246 387 1809 052 445726 884 296 455 1525 646 452285 363 300 328 1890 666 567819 949 301 280 1827 193 550496 703 376 369 4000 729 1505750 327 Using Auto Experiment Results viewer Chapter 15 Automated operation Displaying spectrum and Mascot data Nodes that allow you to display their results as either a spectrum or Mascot results have a grey square at its top lef
21. Off Closed Reflectron Reflectron Reflectron a Fail Fail Fail X Defl Y Defl Detector eect Figure 11 5 Axima Performance status diagram The key at the bottom of the diagram indicates the possible states of the unit Axima Performance instrument status Chapter 11 Checking the instrument status Axima Performance status diagrams key The items on the diagram and their possible states are described in the table below Table 11 2 Axima Performance status diagram key Item Status Explanation Laser Off On The laser is shown in the Off colour blue when not ready to fire and in the On colour green when ready Rotary Always on This pump is used to pump the pump no inlet chamber from atmosphere indication and to pump the exhaust from colours the turbo pumps Turbo Fail Off The pumps provide the high pumps Accelerating vacuum They are only or At speed switched off when the instrument is vented The turbo pumps are shown accelerating when the blades are running at less that 80 of full speed Vacuum Fail Poor The gauges are always on The gauges OK gauges reads pressure in Millibar Pascal and or Torr units via a context menu over the window HT supplies Fail Off On The supplies are switched on when the instrument is fully pumped and enabled see Preparation for data collection on page 117 They are always switched off before the door is opened V1 Open This valve isolat
22. Prediction of Protein Antigenic Determinants from Amino Acid Sequences Proc Natl Acad Sci U S A Vol 78 Pp 3824 3828 1981 Johnson R S and Biemann K Computer Program SEQPEP to Aid in the Interpretation of High energy Collision Tandem Mass Spectra of Peptides Biomed Environ Mass Spectrom Vol 18 Pp 945 957 1989 Johnson R S Martin S A and Biemann K Collision Induced Fragmentation of M H lons of Peptides Side Chain Specific Sequence lons Int J Mass Spectrom lon Processes Vol 86 Pp 137 154 1988 Kyte J and Doolittle R F A Simple Method for Displaying the Hydropathic Character of a Protein J Mol Biol Vol 157 Pp 105 132 1982 Roepstorff P and Fohlman J Proposal for a Common Nomenclature for Sequence lons in Mass Spectra of Peptides Biomed Mass Spectrom Vol 11 No 11 Pg 601 1984 Stults J T and Watson J T Identification of a New Type of Fragment Ion in the Collisional Activation Spectra of Peptides Allows Leucine Isoleucine Differentiation Biomed Environ Mass Spectrom Vol 14 Pp 583 586 1987 Introduction 641 Chapter 42 Sequence Calculator 642 Introduction Chapter 43 Listing of the template itn file Chapter 43 Listing of the template itn file 643 644 Chapter 43 Listing of the template itn file ead Internet Search Information File ae Version Revision 1 8 Locker ake 2000 09 13
23. 100 50 0 h LLL Ye Pe ILS LM LK 500 1000 1500 2000 Figure 20 34 Adjusting the base height in Isometric plots Customising Graphical Reports 379 380 Chapter 20 Managing Data Displays Customising Text Reports The Text Report property page Figure 20 35 allows each text report display to be individually styled Display Options BBE General Graphs Graph Text Text Report Cursors Peak Labels Section headings IV Blank after section headings I Column headings M Blank after column headings Lines to display 5o 4 Lines to print es Al displays ok _ amy cance Figure 20 35 Text Report property page The Section headings option selects whether or not the section heading of a text report appears The heading is that part of the report which appears at the top of each page above any column headings see Graph Headings on page 373 The style of the title will depend on the text report currently displayed The Column Headings option can be used to enable or disable the column headings The Blank after section heading option can be used to add a blank line after the section heading and similarly Blank after column heading adds a blank line after the column heading between the column headings and the information Customising Text Reports Chapter 20 Managing Data Displays The Lines to display option allows the number of lines displayed on a page to be controlled Decreasing the
24. 213 214 Chapter 15 Automated operation ASCII Text Method file format The RunPlate method file facility allows the definition of a complete experiment The parameters for the run plate method are an ASCII based stream that is HTML like format It is block structured and each block starts with the block name and ends in the negation of the block Parameters are single line based as defined below lt BlockName gt lt BlockName gt lt ParameterValue Value inside double quotes gt All block names and parameter value names are case insensitive Whitespace in the stream is stripped and ignored Whitespace is defined as space tab carriage return and line feed characters The currently defined blocks and parameters are as follows lt Experiment gt lt Plate gt lt Title string gt lt Width float mm gt lt Height float mm gt lt Well gt lt ID string gt lt X float mm gt lt Y float mm gt lt Geometry ordinal gt circular square rectangular lt Xsize float mm gt diameter for circular lt Ysize float mm gt lt Well gt lt Plate gt lt Group gt lt Well gt lt ID string gt Must match a well defined in the plate definition lt Comment string gt lt Well gt lt Method gt lt DataStorage gt lt DataStorage gt lt MS1 gt ASCII Text Method file format Chapter 15 Automated operation lt Acquisition gt lt Raster gt lt AutoQuality gt lt LaserFiri
25. Annotation _terly_ Remove een seca Fit through zero Polymers heals Disses levee kil d se Seine ead R Type E Z Dataset A datasets zl fo HE Reference Editor Trace AI Traces w Sort Increasing mass X a Select Peaks Dataset Trace ali dha Combined Cal Peak Processing BBE yO 19 02 b 17 1 99 10 i1 8809 Dataset E 1 ang11_0001 7 rae F Sampe 02 y1 166 b 17 2 255 28 Peak Cleanup Peak Picking Peak Filtering 2129 19 Manual Peak Assignment Internet Search 4B Chromatography y soe Advanced Detection Advanced Settings Hi Dataset 1 pep_mix_2466d0005 Profile average ll profiles Tagged profiles Mass range 3000 12000 2 Te Peak width 2 Shans F Peak area G pan c Tak Smoothing method Average gt Method Gradient Smoothing Average Eee e a pmen Segments 20 I Combine Smoothing fiter width 20 chans i Peak se Peakheight 2 mv Subtract baseline Detect peaks Baseine fiter width 60 chans Peaks detected Peak detection method Threshold 25 Centroid z ia a Po Both Threshold 25 Centroid Peak Detection Settings Double Threshold r Hie Threshold type C piiras e piling Threshold offset oso mv m Threshold response 1 000 x my Apex mV Profiles 101 250 11250 11549 751 1000 11100 11549 1301 1450 11100 11549 1651 1900 11100 11699 eee toply to Ttes
26. Headings Camera X Axis V YaAxis V Display Heading IV oieee x Aris Title V Y Axis Title V Trace Headings IV Database Viewer Tile Manager Separate X Axes I Event Filters r Markers Baseline V Peak Limits I Threshold V r Scaling Profile Scale Automatic x Isometric plots Angle as 4 Base height 50 4 r Colour editors Colour Editor buttons Spectum Chromatogram Distribution Comman P Alldisplays Figure 24 1 Display Options Colour Editors The Graphs property panel allows the colours to be edited for displays having a graphical content These are namely Spectrum Chromatogram and Distribution Items common to all graphs e g axis colours etc are edited using the Common colour editor 430 Introduction Chapter 24 Choosing user defined colour schemes Changing spectrum trace colours There can be charged traces and neutral n traces for each of the 10 loaded datasets the colour of each of these traces can be set independently Select Spectrum from the available colour editors the window shown in Figure 24 2 will be displayed W colour Editor Spectrum Traces BE Traces Miscellaneous naisi ama em ee Baseline Colour sets a Load Load defaults EA Peak limits Apply Figure 24 2 Colour Editor for Spectrum traces To change the colour for a particular trace click the mouse
27. Min points 5 Auto calibration Performance measurement options C Abort experiment C Prompt for user s intervention Lock Mass Calibration 7 Lock Mass p 0000 Tet Tolerance jo Da z C Skip to next sample Save Method Figure 15 7 Method Editor Auto Quality Load Method Method Editor 171 172 Chapter 15 Automated operation Laser Firing The Method Editor Laser Firing window is similar to that described in Collecting data from a sample on page 139 To access the Laser Firing window select the Laser Firing label from the tree as shown in Figure 15 8 for Axima Resonance QIT instruments Figure 15 9 amp Figure 15 10 Method Editor default mtd 1 x Sample Method Sample Method default mtd Parent Laser Firing Vy Parent Ge My Acquisition Setup Vv Ea Raster viv X Auto Quality Configuration defautteflecton TA J M Processing Mass Range 1 0 10000 0 VIL Calibration EZ Peak Cleanup Calibration nd TIJ Monoisotopic Peak Picker v EN Peak Filtering VIS Mascot Applications p Laser Firing _ Compare Sequence Oligo Analysis Power 90 MQ Output ei Profiles 10 Sj per sample n J Report amp lon Finder Shots 100 v accumulated per profile VS MS MS GMI Acquisition Laser Rep Rate 50 0 Peak Selection AA Raster IonGate Off on Blank 500 0 700 0 el LMZ v 3E Laser Firing TMa EA Cleanup M P Ext optimise for
28. Os a co saseag fa oon to adds CJ Gants Simulated resolution 2000 a z Mass List Contents SBE Trace Sample SB emes a Show Mass Area Apex mV Total Resolution Signal Noise Flags Precision 2 decimals gt Maximum listed peaks 50 i Minimum peak apex 0 000 x fa Significant peaks only I suomesoe ou ia t Calibration Contents Dataset Trace Sample i T angio2_msms0l_ Fi er a e a Reference Contents BBE Data fror SERENE Reterence Traces Profile Peaks Auto mass range V Resolution 2000 Figure 5 22 Display contents windows a Display contents windows Chapter 5 Window and menu guides 3 Reference List Contents Data from E fg Calibrant List Contents SBE SBE ba Notes Contents Dataset L is Note Spectrum te Derbuten BREE Calibration Reference Browse Edit Delete Polymer Statistics Polymer Graph Folder C Program Files Shimadzu Biotech of Mass List File angio2_msms0006 note1 Reference List Calibrant List Notes Summary Sequence Calculator Results Ganons Peak Match p gt E Summary Contents BE Instrument Record Information Auto Experiment Results PRESSE Peptide Mass Fingerprint Results ore onj Mascot Search Results Intensity Mapping 2 2 plr mems0004 2 plr mems00
29. Read failed for element number of isotopes value Read failed for element atomic number value Read failed for element min DBE value Read failed for element max DBE value Read failed for element average mass value Read failed for element padding value Read failed during element value Retry loading the file the file may have been corrupted or may contain invalid information If this is the case either copy the original periodic_table data file into the databases directory or restore a previous version from a backup Error 16150 Error 16160 Error 16170 Error 16180 Error 16190 Error 16200 Error 16210 Error 16220 Error 16230 Error 16240 Error 16250 Error 16260 Error 16270 Error 16280 Error 16290 Write failed for element mass value Write failed for element abundance value Write failed for element specific gravity value Write failed for element melting point value Write failed for element boiling point value Write failed for element conductivity value Write failed for element symbol value Write failed for element full name value Write failed for element number of isotopes value Write failed for element atomic number value Write failed for element min DBE value Write failed for element max DBE value Write failed for element average mass value Write failed for element padding value Write failed during element value Retry after c
30. Sr My Computer amp My Network Places Save in e Peptides x e Fe E ACTH 1 17 ksq Sl ACTH ksq E angiotensin 1 ksq E angiotensin 2 ksq E angiotensinogen ksq E Bil test ksq E default ksq E insulin b ksq E pl4r ksq E standards ksq Save as type Sequence files ksq x Cancel Figure 42 20 Save Sequence window Sequence reports Reports are generated from the displayed sequence in the selected viewing panel of the Sequence Calculator window An amino acid or peptide sequence can be modified by the addition of different N and C terminus and cation groups The peptide can be digested using specific enzymes and a variety of fragmentation information can be provided including mass 620 Introduction Chapter 42 Sequence Calculator spectral and multiply charged fragmentation All of these options are selected using the Fragmentation tab of Peptide Settings see Figure 42 21 below Peptide Settings Keyboard Display Fragmentation Match Peaks Nested PSD Spectra Digest fragments lonisation fragments Singly charged N Terminus C Terminus Report Fragment number Vv _a ar a2 217 a18 _x ef asses v Bull Breese indices MV mafea _2 z HPLC indices M E fifa shele Elemental Formulae V Sequence M Clear Method Trypsin X Sort by Mass Charge Missed cleavages 0 Musee cherned Mass imits 0 2000 e
31. on page 293 Chapter 17 Viewing the collected data Displaying Spectra Set Display to Spectrum Spectra are the standard displays of signal intensity against mass Figure 17 3 Four types of spectral traces are available individually or in any combination these are summarised in Table 17 2 Table 17 2 Traces for spectrum Trace Data drawn Profile Displays data collected for each profile from the selected sample Averaged Displays the average of all the profiles from the selected sample When using a continuous slide this displays the last set of samples averaged Processed Displays the averaged data after the application of smoothing baseline subtraction and peak detection to the data if requested Peaks Displays the centroided apex mass peaks found in the processed data The four different traces Figure 17 3 are selected in the Display contents window Figure 17 2 available from the View menu Spectrum Contents Dataset 1 angl1__0001 Sample Process 2 psd_angl1__0001 Scroll dataset Profil A Pi Peak faces tone verage tocess eaks K View Stack Overlay Set 15 610 Display multiple samples I Displaying Spectra 269 270 Chapter 17 Viewing the collected data Figure 17 2 Spectrum contents window olnt 980 mY Profile 54 Profile 100 50 0 lnt 980 m sum 52915 mW Profiles 1 54 Averaged Average 100 50 E lnt 480 m sum 25928 mv Profile
32. therefore the Generate Spectra button changes to Cancel during operation so that it is possible to abandon the process at any stage The colours used to display individual ionisation fragments are shown in the table Table 42 5 Colours of Peptide Fragments Fragment Ion Colour RGB Values A Red 255 0 0 A Pink 255 192 203 At Orchid 218 112 214 A 17 Moccasin 255 228 181 B Blue 0 0 255 B Turquoise 64 224 208 B Cyan 0 255 255 B 17 Misty Rose 255 228 225 C Green 0 255 0 GC LimeGreen 50 205 50 cm ForestGreen 34 139 34 Da SlateGrey 112 128 1440 Db Grey 190 190 190 l FireBrick 178 34 34 M Tan 210 180 140 Introduction 633 634 Chapter 42 Sequence Calculator Table 42 5 Colours of Peptide Fragments Continued Fragment Ion Colour RGB Values X Yellow 255 255 0 x GoldenRod 218 165 32 xe Khaki 240 230 140 Y Orange 255 140 0 Yi Salmon 250 128 114 Nees Brown 165 42 42 Z Purple 160 32 240 Z SkyBlue 135 206 235 Zii Magenta 255 0 255 V Maroon 176 48 96 Wa NavyBlue 0 0 128 Wb DodgerBlue 30 144 255 Sequencing using Nested PSD Introduction The method of nested PSD post source decay can be useful in determining the amino acid sequence in a peptide chain The basic technique is to treat a peptide with an enzyme or an alternative chemical work up which selectively cleaves one or more amino acids from only
33. 1500 0 w v TA Monoisotopic Peak Picker Restrain peak cleanup MILES Peak Filtering VIS Mascot Wy Output x gt Method Editor Data Storage Get Manual Settings Save Method Load Method Figure 15 8 Method Editor Laser Firing The mode of the instrument can be defined exclusively for automated acquisition by selecting a previously saved tuning file see Preparation for data collection on page 117 from the Tuning mode drop down list Set the mass range of the instrument during automated acquisition in the Mass Range field See Preparation for data collection on page 117 Define the calibration to use for the current acquisition by selecting a previously saved calibration file from the Calibration drop down list Chapter 15 Automated operation Set the laser power as described in Collecting data from a sample on page 139 Enter the number of profiles per sample as described in Collecting data from a sample on page 139 Set the shots accumulated per profile as described in Collecting data from a sample on page 139 Method Editor default mtd x Sample Method Sample Method default mtd QIT Laser Firing Vig MS Wy Acquisition v fa Raster Mx Auto Quality Tuning mode positive x VY Laser Firing QIT WEY Processing Mass Range fico 10000 0 MLE Calibration PA EZ Peak Cleanup Caibratio S VIIN Monoisotopic Peak Picker VEA Peak Filtering vS Mascot W Appli
34. 1530 1540 Mass Charge For Help press F1 IdM 135 13 MjdM 3 29 NUM Figure 20 49 Linked displays If the mass range of the spectrum is now changed e g expanded by dragging the mouse the mass list will also update to display the peaks within the displayed mass range The selected display must contain a spectrum or chromatogram to enable other displays to be linked to it links may not be created to a text report When a linked display is no longer required move the mouse pointer over the linked display and press Alt with the mouse SELECT button the green border will be removed and that display will be unlinked from any others The original display the display to which the others are linked is shown with a cyan coloured border the displays linked to it are always shown with a green border Any number of displays may Linking data displays 399 Chapter 20 Managing Data Displays be linked to a single display so that several displays are updated together Each time a new data display is linked it is shown with green borders To remove all links to the original display press Alt with the mouse SELECT button over the original display All of the green borders will be removed from linked displays and the displays will be unlinked The type of link obtained depends on the data range on the X axis of the selected display s graph For instance if a link is made to a chromatogram the shot range of the linked display can
35. 250 Resolution Standard Resolution CID Control CID Control Soft hard o Adjust Gate Clear All Apply 498 Checking the ion gate Chapter 29 lon gate calibration a Enter the Precursor lon mass 1000 90 b Select the required Resolution Std Isotopic Selection 250 Set CID Control to O i e off d Click the Apply button 9 Go to the Firing window acquire a spectrum and zoom in on the peaks 10 Click the Suspend button 11 Measure the boundaries of the ion gate i e where the peaks stop start see example on the next page a Click the right mouse button and move the cursor to one of the boundaries b Note the mass in the bottom left display c Repeat for the other boundary 12 Calculate the mid point between the boundaries and compare it with the Precursor lon mass set in the QIT ToF MS window In the example below the ion gate is 4 2 Da wide The mid point of the ion gate is 1000 90 Da The precursor mass was 1000 88 Da i e 0 02 Da error This error is not significant and the ion gate is operating as expected A 6 lt Untitled gt MALDI MS File Edit View Instrument Automation Processing Help i m B aj al Display spectrum oe Profis z Messes 987 1007 J 1491 lnt 26 m sum 5196 mY Profiles 1 200 Unsmoothed 1000 02 1000 99 For Help press F1 1002 1004 1006 dM 5 19 M dM 192 83 NUM Checking t
36. Archiver Enzymes ve Or use the Start menu system Elemente Compounds Petite Programs IT Documentation XG Favorites ta nae 2 Compounds Database Accessories A Documents a a 2 Configure Environment Se fan Administrative Tools amp os gt Settin MGi Shimadzu Biotech Launchpad gs RS Element Database Editor E Y fn Greete a Enzymes 9 Search RoboHelp Office eres 2 g 7 Help and Support SIAMES Log Window SoundMAX BB earns a amp 1 Ha T Symantec Client Security Sample Plate Editor o Log Off bill volo View Express E oe n g Search l a z Turn Off Computer Figure 40 1 Starting the Polymers window Introduction 590 Chapter 40 Polymer simulation The Polymers window will be displayed Figure 40 2 gt Polymers Big gk Help Polymer files Reference nylon nylon Load Save Polarity Generate Mass Intensit Formula 1586 2557 1286 H NH CH2 5 C0 14 H 1699 4157 2387 H NH CH2 5 C0 15 H 1812 5757 6564 H NH CH2 5 C0 16 H 31925 7357 9360 H NH CH2 5 CO 17 H 2038 8957 9820 H NH CH2 5 C0 18 H 2152 0557 7580 H NH CH2 5 C0 19 H H NH CH2 5 C0 20 H Fa gt Polymer details Formula H NH CH2 5 C0 n H Average molecular mass 2000 0000 Daltons Isotopes Mass window 1000 0000 Daltons Average masses x ite 10000 Minimum intensi
37. C9 Stage X a e More 5 96x4700 00 de2111ta plt 5 384x340 4x48 fleximass to 483r00 plt 384x2000 00 de1271ta plt 5 384x340 4x48 quickmass to 484r00 plt 384x2000 00 de2113ta plt ABplate 1 96x3400 00 de1583ta plt 5 384x2800 00 de1580ta plt masstect 5 96x3400 00 de2112ta plt 384x2800 00 de2115ta plt plain de1 gt 96x4700 00 de1487ta plt gt 384x2800 96 de4555ta plt plain dez gt Filename _ 384x2800 96 de4555ta plt Files of type Plate Files plt x Cancel You can set the plate selected here to be the default plate The default plate is displayed at Auto Experiment start up unless it is replaced by a plate loaded from an experiment file The checkbox allows you set the selected plate as the default Save the current plate to a file This feature is useful when a Chemical Printer or free hand experiment has been imported and the user wishes to save the sample positions as an Axima plate Clear all selected wells from the Selection plate Increase the magnification of the Selection Detail This allows easier selection of single wells especially when using higher density plates Decrease the magnification of the Selection Detail This allows easier selection of groups of wells especially when using higher density plates Auto Experiment 205 Chapter 15 Automated operation Table 15 6 Auto experiment plate icons Toggle the Experiment plate view bet
38. Chapter 13 Preparation for data collection To load the plate file associated with the sample plate in the chamber select the Load button and select the relevant file from the list available on the standard load file dialogue bel 384x2800 00 de1580ta plt Sample Plate Editor x Save as Print Help Plate specification 384 2 8mm wells i Plate X size mm 77 00 Plate Y size mm 125 00 Plate x offset mm 9 00 Plate Y offset mm 0 00 Number of sample wells 384 Alignment Refs Auto define wells Individual sample well specifications well index A1 a Well geometry Circular well 7 Well position centre point of well coordinates x mm 72 25 Y mm 114 25 F Well dimensions p Diameter mm 2 80 Append wen Inset well Before Delete Wel Delete All After C Figure 13 6 Sample plate editor window To create a new plate file or edit an existing one select the Edit sample plate button the editor window shown in Figure 13 6 above will appear The well dimensions are provided in millimetres to the centre of the well from the origin which is assumed to be the lower left hand corner of the sample plate where the first well A1 on a standard plate is assumed to be towards the upper right hand corner of the sample plate see Figure 13 8 below Existing wells can be scrolled using the up and down arrow keys adjacent to the Individual sample well specificat
39. Chapter 36 Printing the contents of displays There are two modes of printing from MALDI MS displays If the exact contents of the display are to be printed then select Print from the File menu This will display the Print window as shown in Figure 36 1 Print x Printer Name HP LaserJet 8000 DN PS Properties Status Ready Type HP LaserJet 8000 Series PS Where HP8000DN Comment J Print to file Print range Copies All Number of copies BE c a Cancel Figure 36 1 Print window This window may be different depending upon the type and manufacturer of the printer and the Windows printing system used However in all cases it allows the printer properties print media orientation and scaling to be adjusted Select the number of copies to print and press OK Alternatively to preview the printout in order to check that what will be printed is what is expected select Print Preview from the File menu This will display a preview of the printout within the MALDI MS main window as shown in Figure 36 2 Click on Close when the preview is finished with and the MALDI MS window will return to the normal view If the display contains a text report such as a mass list or other textual information then to print the whole report select Print Text Report from the File menu Chapter 36 Printing the contents of displays nsms0004 MALDI MS EA rare marae Zoom In Zest Close Data p14r_msms0004 4 Ma
40. Comment line text Data angio2_msms0006 G7 c 4 Mar 2005 14 37 Cal tof 4 Mar 2005 14 37 CID of 1046 51 Shimadzu Biotech Kompact MALDI 6 2 7 0 Beta 1 Mode reflectron_ms_ms Power 73 Gate 1035 00 1060 00 P Ext olnt 73 m sum 41728 mW Profiles 1 54 Smooth Av 5 Baseline 80 100 se a Display heading Trace heading 80 60 q Y axis 40 X axis 20 t X axis title o 1041 1042 1043 104 1046 Mass Charge 1047 1048 1049 1050 1051 Figure 20 32 Optional items on a graphical display 374 Customising Graphical Reports Chapter 20 Managing Data Displays Optional items in the display heading Most data displays begin with a standard heading which shows the following components The title for the data as entered in the Comments window The comment for the current sample spot e The name and collection date of the data and its calibration The instrument conditions which applied when the data was collected Figure 20 31 shows a full display heading and graph title on a spectrum report Table 20 5 on page 375 and Table 20 6 on page 376 explains the constituent components of the titles comments and headings in Figure 20 31 Each of these lines can be disabled as required Table 20 5 Display header information Example item Title line text Prefix text Comment line text Data bill angio2_msms G7 c 4 Mar 2005 14 37 Description This is the Title line t
41. Export Headings Profile Average Processed Peaks Format Mass Intensity Report intensities as my T Save as Cancel default Processed data Charged 5807 119 5807 145 5808 177 5809 211 5809 248 5810 284 5810 317 5811 344 5811 362 5812 5813 368 5813 355 5814 333 5814 305 5815 274 5815 241 5816 209 Joe0006 Processed data Charged 5807 26 5807 26 5808 25 5808 25 5809 25 5810 25 5810 26 5811 28 5811 29 5812 31 5812 32 5813 33 5814 34 5814 34 5815 33 5815 32 5816 31 Figure 33 7 Example of a Mass I ntensity pairs export file The Decimal places option is available when writing either Processed data or Mass Intensity pairs to the export file as the masses and processed data contain floating point numbers all other values are integers The Report intensities as mV checkbox if unchecked gives the intensities as counts returned from the instrument Note that the values reported in both cases are actually summed intensities across the peak i e areas and not peak apex intensity values The exported files can be imported into spreadsheets using matching import filters to delimit the entry fields within the exported data Exporting ASCII data 529 530 Chapter 33 Exporting data and data displays Exporting data displays as meta files The Export sub menu in Figure 33 1 above shows two other options as well as exporting the buffered data in ASCII format these options allow data displays thems
42. Introduction The operating mode of the instrument and the method of sample selection are set from options on the Instrument menu on the base window Select Acquisition on the Instrument menu The Axima instrument s Experimental Technique Tabs have a Configuration drop list selector which allows previously saved parameters for a particular experiment to be loaded The window also has radio style buttons to place the instrument in Standby or Operate mode and to set Manual or Automatic door control The mass range of interest can also be set in this window and this governs the number of data sample bins The maximum mass range for the Axima is 1 500 000 but for the Axima Resonance the mass range is limited to approximately 10k Da by the operation of the ion trap though higher masses may be specified in this window The CID button is available on the Axima Performance instrument It provides the ability to switch on a collision gas which can enhance fragment ions produced in post source decay experiments An example of the effect of CID gas is shown Figure 13 1 below where the immonium ion fragments of angiotensin II are increased significantly when the gas is present upper trace This is an Enable Disable toggle button there is no user adjustment of the CID gas pressure If the instrument is in operate the software will automatically switch to standby Then the CID gas valve is opened and the gas pressure allowed to settl
43. No 8 Vol 2 151153 1988 R J Cotter Time of Flight Mass Spectrometry for the Structural Analysis of Biological Molecules Anal Chem Vol 64 No 21 1027A1039A 91992 S Minami Wave Form Data Processing for Scientific Measurements CQ Publishing 84121 1986 J F O Hanlon M Noda Y Saito F Okuya Manual of Vacuum Technology Sangyo Publishing 638 1983 Paizs B and Suhail S Fragmentation pathways of protonated peptides Mass Spectrom Rev 24 508 548 2005 Papayannopoulos IA The interpretation of collision induced dissociation tandem mass spectra of peptides Mass Spectrom Rev 14 1 49 73 1995 Chapter 46 Acknowledgements Chapter 46 Acknowledgements 665 666 Chapter 46 Acknowledgements UNIX is a registered trademark of UNIX Systems Laboratories Inc MSDOS and Windows are registered trademarks of Microsoft Corporation IBM is a registered trademark of International Business Machines PostScript is a registered trademark of Adobe Systems Inc Mascot is a registered trademark of Matrix Science Ltd
44. Profiles 101 250 11250 751 1000 11100 1301 1450 11100 1651 1900 11100 11699 Brevious First Last Clear 4 peak s selected i Peak display ow Figure 30 7 Manual peak detection Tagging peaks using the Chromatography window All of the peaks in the Peaks detected list can be tagged for use in peak cleanup as described in Smoothing collected data on page 240 to improve the signal noise ratio by excluding poor shots with little or no signal To tag the peaks in the list press the Tag peaks button To remove all tagged peaks press the Clear tags button Individual peaks or peak ranges may be tagged or untagged on a chromatogram 2 D plot A 2D plot is selected by setting Segments to 1 in the Chromatogram Contents window and then pressing the Apply button On a 2D plot tagged peaks appear in a different colour to untagged peaks Use a pair of cursors to delimit a peak or a range of peaks then from the menu which appears by pressing and holding down the mouse MENU button select and pull right on the Tags option see Figure 20 1 on page 335 The options Tag and Clear will add or Chromatographic peak detection 512 Chapter 30 Chromatography remove tags respectively to all peaks within the cursor range The Clear all option does not require the use of cursors and removes all tagged peaks associated with the current data in exactly the same manner as the Clear tags butto
45. Resolution 1000 at 50 100 80 60 40 20 0 2160 Mass Charge Figure 23 9 Expanding the reference peaks 426 Displaying reference files Chapter 23 Displaying simulated data The Resolution parameter allows the distribution to be simulated at any instrument resolution Increasing the resolution increases the level of detail seen in the peak profile Figure 23 10 956 52 50 55 90 965 970 Figure 23 10 Increased oe provides increased etai The Auto mass range option has been provided to automatically scale the mass range to that of the lowest and highest masses in the reference mass range This means that the reference display will always include all of the reference peaks in the file If Auto mass range has not been selected then the previously selected mass range will be retained The whole range of reference masses can be obtained by pressing the toolbar Ki full range button The reference display can be scrolled zoomed and manipulated as with all of the other display types Displaying reference files 427 Chapter 23 Displaying simulated data Listing peaks in a reference file To display the masses and formulae of each peak in a reference file set the Display type to Reference list This produces a report with two columns showing Mass and Formula Figure 23 11 mixed_ref0001 MALDI MS olx File Edit Yiew Instrument Automation Processing Help i HES 2j 8
46. Select the Manual peak assignment start icon to open the feature a Manual Peak Assignment Be x J oast 1 8402 0002113 o ener Delete masses Delete gt Selected The peak that you set appears in the manually assigned mass list Deleting a peak Peak 1 Select the pa icon the cursor changes to H Del 2 Move the cursor to the required peak label and click the mouse left button peak label is removed and the peak mass is removed from the Mass list Deleting a range of peaks 1 Position cursors click mouse middle button either side of the range 2 Select the X icon peak labels are removed and the peak masses are removed from the Mass list Peak labelling 4il 412 Chapter 22 Manual peak labelling Manual peak assignment Select Manual Peak Assignment from the Processing menu options on the base window Figure 22 2 angio2_msms0006 p14r_msms0004 MALDI MS Biel Eg File Edit View Instrument Automation Processing Help z Calibration ile T J AE Hag BI 8 x ala Profiles 1 Masses 12 1040 i gt i Peak Processing Chromatography Manual Peak Assignment EG Polymers Sequence Calculator Al Dataset 1 Reference Editor Select mass Select Peaks Sr Manual Peak Assignment Internet Search Delete masses Selected z Figure 22 2 Manual Peak Assignment window Manual peak assignment uses the Spectrum display to delimit peak boun
47. and DE The second level of missed cleavage yields ABC BCD CDE and so on The Missed cleavages option sets the required level permitted in the report A mass limit can be set on the enzymatic digest fragments Fragments of mass above the value of Mass limit are not reported Introduction 623 624 Chapter 42 Sequence Calculator Introduction Having made all of the required selections press the Apply button PSD Fragmentation Matrix assisted laser desorption ionisation MALDI is a well established method of ionising and analysing peptides The mass spectra obtained often contain an abundance of fragment ions which can yield a considerable amount of structural information The rules controlling fragmentation are well understood and it is possible to predict the fragmentation pathways of a specific peptide with a reasonable degree of certainty When the Fragmentation option on the Sequence Calculator window is set to Singly charged the buttons in the I onisation fragments section of the Fragmentation tab allow the user to select which PSD fragment series should be reported The following two tables provide a comparison between the two notations and define the fragment ions The tables use the following definitions N is the mass of the N terminal group C is the mass of the C terminal group M is the mass of the sum of the neutral amino acid residues Table 42 3 N terminus ionisation fragments
48. from the View menu Figure 20 28 E default MALDI MS Oy x File Edit view Instrument Automation Processing Help ket v Toolbar Display Spectrum gt Profiles 1 Masses 12 1040 1 gt wees V Status Bar v Display Toolbar v Basic Parameters Display Contents Camera Instrument Status Database Viewer Tile Manager Event Filters Figure 20 28 Selecting the Display Options The Display Options window has six tabbed property pages General Graphs Annotation Text Report Cursors and Peak Labels Select the Graphs property page Figure 20 29 Customising Graphical Reports 371 372 Chapter 20 Managing Data Displays Display Options Bea General Graphs Graph Text Text Report Cursors Peak Labels Headings X Anis V YAris V Display Heading M X Axis Title V Y Axis Title V Trace Headings M Separate X Axes I Markers Baseline V Peak Limits I Threshold V r Scaling Profile Scale Automatic 7 Isometric plots Angle 145 4 Base height 5o r Colour editors L Spectrum Chromatogram Distribution Comman P All displays Figure 20 29 The Graphs property page On this page the various items which comprise a graphical display such as the main headings graph titles and the X and Y axes can be customised to suit specific requirements The X and Y axes can be shown or hidden along with X and Y axis labels Multip
49. gt lt PrintType enumeration gt Spectra MassList SeqReport Window lt SpectraDataName enumeration gt true false lt Spectral nstName enumeration gt true false lt SpectraCommenttTitle enumeration gt true false lt SpectraFolderName enumeration gt true false lt SpectraBorders enumeration gt true false lt SpectralstComment enumeration gt true false lt MassListSectionHeaders enumeration gt true false lt MassListSectionHeadersBlank enumeration gt true false lt MassListColumnHeadings enumeration gt true false lt MassListColumnHeadingsBlank enumeration gt true false lt MassListShowMass enumeration gt true false lt MassListShowPercentArea enumeration gt true false lt MassListShowPercentTotal enumeration gt true false lt MassListShowApex enumeration gt true false lt MassListShowFlags enumeration gt true false lt MassListLinesPerPage unsigned integer gt lt MassListPrecision enumeration gt 0 1 2 3 4 5 lt MassListMaxPeaks unsigned integer gt lt MassListMinApex unsigned integer gt 0 100 percent lt MassListSignificantOnly enumeration gt true false lt SeqReportSectionHeadings enumeration gt true false lt SeqReportSectionHeadingsBlank enumeration gt true false lt SeqReportColumnHeadings enumeration gt true false lt SeqReportColumnHeadingsBlank enumeration gt true false lt SeqReportLinesPerPage unsigned integer gt
50. lel AES Ld Display Spectrum x Profiles fi X Masses 12 1040 Kid Optional features and Acquisition instrument specific Maintenance items are in red QIT ToF Experiment Figure 14 1 Opening the Acquisition tab dialogue Introduction Chapter 14 Collecting data from a sample Sample selection The Axima laser Firing tab are shown in the following figures Acquisition Firing Exp Tech Auto Quality Storage Side Raster Tuning I Auto quality Power sc a Profiles fio per sample Shots fort x accumulated per profile Accumulate Profiles I Activate Accumulation Figure 14 2 Laser Firing on the Axima Assurance 4 Acquisition BEI Firing Exp Tech Auto Quality Storage Side Raster Tuning I Auto quality n Profiles fio a per sample Shots orf x accumulated per profile Pleven Ie optimise po H E X Accumulate Profiles I Activate Accumulation Figure 14 3 Laser Firing on the Axima Confidence Sample selection EEEE a 142 Chapter 14 Collecting data from a sample Acquisition i x Firing Exp Tech Auto Quality Storage Slide Raster Tuning T Auto quality Power 50 4 A es Hold Profiles og per sample Shots 20 v accumulated per profile Ion Gate Da Ea On aE i low j MV Pulsed Extraction optimised at Da 2300 mm vous Sx gt t 4 Clear data ous AQA AX F Accumula
51. m2XML File mzData File Intensity Mapping as soir Ion Finder Biomap Figure 33 1 Export options on the File menu Exporting ASCII data Chapter 33 Exporting data and data displays The Export ASCII window will be displayed Figure 33 2 Export ASCII BEI File format 7 Columns 10 4 Delimiter comma 7 Decimal places 0 Expat Headings Profile Average Processed Peaks Format Intensity hd Report intensities as m T Save as Cancel Figure 33 2 Export ASCII window All ASCII export files are terminated with the file extension txt Choose the Delimiter which will be written out between each record in the ASCII file Some spreadsheets are quite flexible and permit the use of spaces commas tab characters and the like Select from the five available separators comma space tab or hash As an example of the output expected in the ASCII export file the data shown in Figure 33 3 was exported default Joe0006 lnt 100 5 mV 1 mY _ 5812 89 100 5810 5812 Mass Charge Figure 33 3 Data used in the export ASCII examples Exporting ASCII data 525 Chapter 33 Exporting data and data displays Having set the desired mass range and selected the datasets to display on the Export ASCII window select the number of columns in which the output is to be written e g two data sets delimited mass range single column Figure 33 4 default Processed data Ch
52. on the selected display The cross hairs of the cursors are used as the insertion points for annotations e g the two ends of a line or diagonally opposite corners of a box Table 20 7 shows the function of the buttons on the New Annotation window 1 M Table 20 7 Annotation button functions Function Annotate with a peak marker to label a peak on the display Annotate with text inserted at the mouse cursor position Annotate with boxed text inserted at the mouse cursor position Annotate with a box between the current graph cursors Annotate with a line between the current graph cursors Annotate with underlined text at the current mouse cursor position Annotate with the mass difference between two range cursors Annotate with the resolution between two range cursors Annotate with an arrow between two range cursors Annotate with an arrow between two range cursors containing text Annotate with an arrow between two range cursors containing the mass difference between the cursors Annotation 383 384 Chapter 20 Managing Data Displays Table 20 7 Annotation button functions Continued wl gt BD i out E E Biz Annotation Function Annotate with an arrow between two range cursors containing the resolution between the cursors Annotate with an arrow between two range cursors containing text with lines dropping to the graph baseline Annotate with an
53. profiles up to twenty four by entering 24 Individual profiles can only be viewed if the collected data was written to disk for every profile It may be that the data collection options were set to average ten profiles and write out the data after calculation of the average Under these circumstances requesting a range of profiles of 24 36 would automatically display 20 40 since data is only available for groups of ten profiles and not for individual profiles The program checks to see how the data was stored and offers the closest range to that requested Where data was only written to disk at the end of a run of 200 profiles requesting a range of profiles of 20 36 would display 1 200 as this is the only data available containing the profiles requested Displaying Spectra Chapter 17 Viewing the collected data Typing in an incorrect range of profiles can be undone by pressing the Undo button on the toolbar aj This will return the profile range to its previous setting The All profiles button gt offers a quick method of displaying all of the available profiles tor the selected sample Choosing the mass range to display The mass range of the displayed data can be selected by typing the required range into the Mass entry on the base window The hyphen can be used in the same manner as for the range of profiles for example entering 100 1000 to plot mass 100 to 1000 The All masses button j displays
54. which is often the case due to some degree of amino acid modification taking place in the peptide resulting in peaks differing only by a very small mass To detect Monoisotopic peaks tick the Monoisotopic check box at the bottom of the Peak Cleanup window and select the Monoisotopic picking tab as shown in Figure 16 11 The default parameters which govern the Poisson modelling of isotopic peaks should prove adequate in most cases These parameters are e Minimum and maximum mass specify the mass range within which to search for monoisotopic masses These default to 600 3500 Daltons at higher masses the instrument resolution can decrease to a level where it may be insufficient to allow the detection of the small monoisotopic peak Though it has been found to be successful up to 6000 Daltons which will in most cases be quite suitable Chapter 16 Cleaning up data MhPeak Processing oy x Dataset IE 1 M5 Ang2 DHB_0001 Trace 77 Sample D9 4 Peak Cleanup Peak Picking Peak Filtering M Use peak picking Method Poisson peptide Minimum mass 1000 5 Maximum mass 3500 4 Minimum isotopes 2 Maximum intensity variation 80 IV Overlapping distributions Minimum peak percent 30 Apply to Tiles Lull je Apply to Samples EI E ml T Advanced Poisson Figure 16 11 Monoisotopic picking tab window Method Poission peptide The software uses an algorithm to pick the monoisotopic peaks Br
55. x Show on startup V APE MALDI MS Search Archiver Enzymes SAL Elements Compounds Polymers 2 The Event Viewer is started g Event Yiewer j 10 x File Action View Help e gt lm 2 Event Viewer Local Application 2 127 event s na QDinformation 12 07 2007 14 18 48 fu Internet Explorer information 12 07 2007 14 18 48 G Information 12 07 2007 14 18 48 Information 12 07 2007 14 18 48 Information 12 07 2007 14 18 48 G Information 12 07 2007 14 18 48 Information 12 07 2007 13 45 46 Information 12 07 2007 13 45 46 error 12 07 2007 12 52 35 Information 12 07 2007 12 45 46 G Information 12 07 2007 12 45 46 Figure 7 1 Event Viewer window 3 Select the required event category Application Security or System 68 Windows event viewer Chapter 7 The Log Window 4 Double click an entry of interest Event Date 12 07 2007 Source MALDI MS Time 09 45 52 Category None Type Information EventID 1 User BILL STEVENS bill Computer BILL STEVENS els Description Could not retrieve configuration settings from the mascot server Check Mascot Setup in the Configuration Editor Data Bytes C Figure 7 2 Event Properties window The Event Properties window is useful for scrolling through messages using the up or down arrow keys The Axima software is capable of generating five categories of message How you set these is described in the next section Wi
56. 09 12 19 o C 1999 Kratos Analytical Ltd Format of entries The entries are in the following format KOMPACT NAME gt lt WEB PAGE NAME gt lt MATCH LIST gt where lt KOMPACT NAME gt is the name used by kompact for an item lt WEB PAGE NAME gt is the name used on the Web page in the HTML source for the same item lt MATCH LIST gt is either which indicates that the program automatically inserts values into the web page or lt KOMPACT VAL 1 gt lt PAGE VAL 1 gt lt KOMPACT VAL 2 gt lt PAGE VAL 2 gt etc which is a separated list where the LHS is the value of the object within Kompact and the RHS is the corresponding value of the same object on the Web page e g Kompact may call an enzyme Arg C but the page lists that as Arginine C Another entry format is HHH HHH HHH HHH HH HH H FIXED lt WEB PAGE NAME gt PAGE VALUE gt where FIXED is a keyword lt WEB PAGE NAME gt is the name of the obj ect on the Web page in the HTML source and lt PAGE VALUE gt isthe value that Kompact should insert into the web page This second format is useful for Web page items which have nothing corresponding to them within Komapct To complete this file for a given database replace the lt gt entries with relevant values from the HTML source Note remove the angled brackets as well Web Address of database lt WEB ADDR
57. 2004_11_18 ANAG 0002_B2 mzXML Generating c temp dave 2004_11_18 ANAG_0002_C2 mzXML Generating c temp dave 2004_11_18 ANAG_0002_D2 mzXML OK Using the command line editor Chapter 34 Batch processor XML export 2006 10 23 14 20 C Data linear test 2004_11_18 ANAG 0004 run Initialising Genera Genera Genera Genera Genera Genera OK ting ting ting ting ting ting Q iF a 0 A Bl C1 D1 B2 L engine temp dave 2004_11_18 ANAG_0004 temp dave 2004_11_18 ANAG_0004 temp dave 2004_11_ 18 ANAG 0004 temp dave 2004_11_18 ANAG_0004 temp dave 2004_11_18 ANAG 0004 temp dave 2004_11_18 ANAG_ 0004 C2 D2 2006 10 23 14 22 Batch Process completed Using the command line editor mzXxMI mzXxMI mzxM mzxMI mzxXM mzXMI 555 Chapter 34 Batch processor XML export 556 Using the command line editor Chapter 35 Using the clipboard Chapter 35 Using the clipboard 557 558 Chapter 35 Using the clipboard The clipboard is a standard feature of Microsoft Windows A clipboard is used by printers during type setting and printer s terminology is retained in Windows for similar tasks so documents or parts of them may be cut or copied on to the clipboard from one application and pasted from it into another In MALDI MS tables such as a Mass list may be copied to the clipboard as ASCII text g
58. 25 Automatic graph labelling scaling and printing Introduction On the base window set the Display type to Spectrum from the View menu select Options Figure 25 1 J default MALDI MS lolx File Edit view Instrument Automation Processing Help v Toobar a4 Display Spectrum Profiles 1 Masses 12 1040 1 gt eee V Status Bar v Display Toolbar v Basic Parameters Display Options 1 x Display Contents Options General Graphs Graph Text Text Report Cursors Peak Labels m Acquisition Print Off he Display ere Instrument Status Database Viewer Pre Tile Manager Event Filters Type Colour x Text scaling 100 Margins mm In C Left fo Top jo Right jo Bottom jo m Headings Display data and cal IV Display folder I Display instrument IV Display borders IV Display title IV Display 1st comment IV Show Auto labels Peak Markers Manual labels Sequence panel colours I All displays OK Cancel Figure 25 1 Display Options window General property page This window s General property page contains parameters which affect the manner in which the graphs are updated and the information displayed on them 442 Introduction Chapter 25 Automatic graph labelling scaling and printing General display options Automatic displays of collected data When data
59. 374 e ifthe Headings Display Title option is ticked then the title for the data is shown as entered in the Comments window General display options 443 444 Chapter 25 Automatic graph labelling scaling and printing General display options If the Headings Display 1st Comment option is ticked then the 1st comment for the current sample spot is shown as entered in the Comments window If no sample comments have been entered then the sample comment line is omitted If the Headings Display Data and Cal option is ticked then the data name and calibration name are shown otherwise they are omitted If the Headings Display I nstrument option is ticked then the instrument conditions are shown e g polarity or otherwise this line is omitted If the Headings Display folder option is ticked then the folder name in which the data is stored is shown in the heading as well as the dataset name If the Headings Display Borders option is ticked then the selected display with a border highlight will be printed with a highlight border on any printouts Otherwise borders are not shown on printed copies Chapter 25 Automatic graph labelling scaling and printing Markers displayed on a spectrum On the Graphs tab there are markers that allow you to switch on off various markers displayed on the spectrum Display Options BE General Graphs Graph Text Text Report Cursors Peak Labels
60. 51 Display toolbar at bottom 1044 m 1050 1052 1054 1056 IIc Mass Charge zal gt gt AP S mm em BB GT ws a0 P For Help press F1 Figure 20 3 angio2_msms0006 MALDI MS File Edit Yiew Instrument Automation Processing Help Ss fia 2j aj x ak Display Spectrum Pr OLX Data angio2_msms0006 G c 4 Mar 2005 14 37 Cal tof 4 Mar 2005 14 37 CID Shimadzu Biotech Kompact MALDI 6 V2 7 0 Beta 1 Mode reflectron_ms_ms F lnt 480 mV sum 25928 mV Profiles 1 54 Smooth Av 20 1046 51 100 isplay toolbar a at left side 1047 53 048 53 049 55 1050 1055 I c Mass Charge For Help press F1 Docking the display toolbar Additional facilities are provided by holding down the keyboard Shift or Ctrl keys when the toolbar buttons are pressed All of the functions of the toolbar buttons are given in Table 20 2 on page 338 and Table 20 3 on page 340 Table 20 2 Display toolbar functions for graphical displays Normal action Copy the contents of the selected display into another Shift Shift Ctrl Ctrl Make an inset of the selected display between the cursors Zoom the selected display to the full window width Zoom the selected display to the full window height The Display Toolbar Zoom to 1 1 x current width Zoom to 1 1 x current height Reduce to 273 current width Reduce to 273 current height fe le
61. 8 profiles are not up to the requirements then an attempt will be made to make up the shortfall at a successful raster point No prescan Here the approach is to start at the beginning of the raster and keep firing at the first point until the quality fails then move on to the next raster point It is thus likely that only a fraction of the rastered points will be fired at indeed perhaps only the first one Data quality The definition of a good datum is based on the values of the resolution signal intensity or signal to noise fields If any of these are set to zero then they are ignored in the subsequent analysis If the profile is not up to the signal to noise requirement it is discarded then the laser power is increased and another attempt is made If this proves to meet the signal to noise requirement then the resolution requirement is tested If the resolution check fails then again the profile is discarded the laser power is Automated data quality filtering eooecene al UJ 154 Chapter 14 Collecting data from a sample decreased and another profile acquired If both tests are passed then the profile is stored and further profiles are acquired and stored until the signal to noise ratio falls below a specified percentage of that of the first accepted profile At this point the next raster spot is selected and the process continued until all the requested profiles have been acquired Using the Auto Quality Software
62. Bl Ne LE disvley Spectun Profis lnt 12 mY sum 627 m Profiles 1 54 Smooth Av 5 Baseline 80 xo 109 96 506 30 617 40 1029 759 20 931 59 103 599 56 919 56 103p op 10 521 16 PAN u i chmod prs A eh dui lanli h 400 500 600 700 800 900 Mass Charge For Help press F1 IdM 151 10 M dM 5 07 NUM Figure 20 50 Using amp inanon to highlight regions of ow signa Amplification Chapter 21 Compound Database Viewer Chapter 2 Compound Database Viewer 403 Chapter 21 Compound Database Viewer The Compound Database Viewer can be useful for identifying peaks or species corresponding to the mass difference between peaks This option is found on the View menu Figure 21 1 F pi4r_msms0004 MALDI MS olx Fie Edit view Instrument Automation Processing Help elo v Toolbar P fad Display Spectrum Profiles fi F Masses 12 1 040 I gt i eee V Status Bar v Display Toolbar v Basic Parameters Compound Database Viewer 1 x Display Contents B Dataset 1 lt Untitled gt Options Category Amino acid x Annotate mass V Camera Instrument Status Report Masses as Average x Decimals None Database Viewer Cursor mass Tile Manager lvycine G ly 6 lanine la Event Filters Usliwn Yn U Cursor mass range Figure 21 1 Compound Database Viewer The lists are comprised of the compounds present in the Compound Database an
63. Calibrant reference files on page 461 Select the default calibration for the acquisition in the Output Calibration section Upon saving the current Method parameters any references in the list will be saved to a reference file and Method Editor 175 Chapter 15 Automated operation associated with the Method This reference file is saved to the default references path and named after the Method e g C Program Files Shimadzu Biotech Launchpad references default pos_ref Sample calibration is then applied as described in Instrument Calibration on page 459 Peak Cleanup The Method Editor Peak Cleanup window provides functionality for cleaning up data To access the Peak Cleanup window select the Peak Cleanup label from the tree as shown in Figure 15 12 below Method Editor default mtd Of x Sample Method Sample Method default mtd Parent Peak Cleanup iy Parent El MY Acquisition v fa Raster Y Scenario Advanced x viv X Auto Quality v 3 Laser Firing Advanced Settings VI Processing YL Calibration Peak width 20 chans ipl EE eak Cleanu Ml Monoisotopic Peak Picker Peak area g AY C gs VIER Peak Filtering k MRR Mascot Smoothing method Average X J Applications J Compare Sequence Smoothing filter width 20 4 chans T Oligo Analysis AZ Output M Subtract baseline V Print 3 Export Baseline Filter width 80 a chans t if OW Report a lon Finder Peak detection method Gradient Centr
64. Chapter 20 Managing Data Displays However several full height displays or several full width displays may be created Figure 20 8 shows the two steps required to enlarge a display ee ee ord BAMEN Bob IOASAS IAD IS VE D We 1 tee meci ne Poser oh Baran i DDB Ste Arava tenn Petes trae Aa amera MD o ee O tet IE ee EOE mmen ma DIa VSI O b maae winen homer ah Brans Bi Te oni Stapam IDa Petes 1162 teen a D bwat 0 EE 9 O fe TER ON a ty TE mer ANETA tom ace meanman boner oh Ravens Mi role aja 1 Select a display 2 Press the display toolbar button aTe eE EA E The display enlarges LCA MCOE T KaU E Jo m to full width e N Figure 20 8 Enlarging a display to full width The same steps are used to obtain all of the enlarged views full width full height and full screen To return an enlarged display back to its original size simply select the required display and press 346 Multiple displays Chapter 20 Managing Data Displays When there are multiple displays and one of the displays is currently zoomed to full screen size the user can step through the other displays zooming each in turn to full screen by using the zoom next display feature Pressing the toolbar button will show the next display in the series zoomed to full screen Each display can be zoomed to full size in turn After the last display in the sequence has been zoomed the seq
65. Delete option on the Manual Peak Assignment window In the peak list highlight all of the peaks to be deleted using the Manual peak assignment 413 414 Chapter 22 Manual peak labelling mouse SELECT button and choose Selected from the Delete gt options Alternatively choose All to delete all of the manually assigned peaks Manually assigned peaks are flagged as M Manually assigned in the mass list report Manual peak assignment Chapter 23 Displaying simulated data Chapter 23 Displaying simulated data 416 Chapter 23 Displaying simulated data Introduction The following data displays do not show collected data Instead they are used to simulate the expected profiles of isotopic distributions within collected data and can therefore be used to compare actual data with theoretical predictions Introduction Chapter 23 Displaying simulated data Displaying isotopic distributions Distribution displays are used to simulate the theoretical isotope distribution for any molecular species whose elemental composition is known It is possible to display the peak profiles which will appear with a given instrument resolution With this tool complex macromolecules can be simulated and the expected peak shapes predicted This can certainly be of assistance in locating fragment ions and parent molecules within the mass spectrum To create a distribution display set the Display option to Distribution then click
66. Error 24130 Memory not available for creation of a baseline buffer In all of the above cases close other programs shutdown active processes to free more memory if the problem persists Shutdown Windows and reboot Error 25000 Unknown database format The requested sequence database is in an unsupported format the only option may be to copy the sequence into a text file and use the Import feature of the sequence calculator Error 25010 Error mapping the database file filename Error 25020 Error creating the database index file filename Error 25030 Error mapping the database index file filename Retry after checking file and directory write permissions on the databases directory being written to Check the available disk space in the directory being used If nothing is obvious the specified file may have been corrupted and contain invalid data Error messages 659 660 Chapter 44 Summary of error messages m Warning messages Warning 1000 If data is written into this directory the archiver will not be able to see it To ensure data can be archived save relative to folder Data can be stored here but it is recommended to put the data in a subfolder of folder The Archiver will only look in the data folder registered with the Configuration Editor Warning 1010 Mode set to Standby Set to Operate to continue The operation of the laser has been disabled by setting Mode Standby Data collection will not occur until Oper
67. Figure 17 7 Display contents window for mass lists Select the dataset for which the mass list is required along with the Trace and Sample Select the columns to be shown in the mass list report by using the left and right arrow button on the Mass List Contents window to show or hide the various columns The masses listed can be shown with up to 5 decimal places as selected by Precision The Mass List Contents window has several features to restrict the number of peaks listed The peaks listed in the report are also limited by the Mass limits entered on the base window as for spectra The mass limits can also be changed by choosing a range from another display which contains a spectrum or by selecting a range from the graph as explained in Choosing the mass range to display on page 273 Producing a table of peaks in a spectrum Chapter 17 Viewing the collected data J pep_mix_2466d0005 MALDI MS ojx Eile Edit view Instrument Processing Help Sli 8 8 2 S 2 e cv PRERE Data pep_mix_2466d0005 11 Ofc 10 Feb 2005 10 06 Cal tof 10 Feb 2005 9 02 Shimadzu Biotech Kompact MALDI 6 2 7 0 Beta 1 Mode reflectron_ms Power 45 Blanked P Ext 2460 bin 91 Mass Area Total Apex m Resolution S N Flags 1001 85 0 12 0 02 0 01 2710 19 9 69 1002 82 0 20 0 04 0 02 2857 28 18 36 1004 11 0 12 0 02 0 02 2432 41 11 27 1006 29 0 21 0 04 0 02 2895 11 14 54 1014 83 0 08 0 02 0 01 2934 35 6 88 1016 93 0 08 0 02 0 01 29
68. For Help press F1 dM 135 13 MidM 3 29 Figure 20 47 Panning the mass range using two displays 396 Panning displays Chapter 20 Managing Data Displays If the selected display is a spectrum and the mouse is currently within a chromatogram display pressing Ctrl with the mouse SELECT button over the chromatogram and moving the pointer will show the spectrum in the selected display for the profile under the mouse Figure 20 48 J pep_mix_2466d0005 MALDI MS ojx File Edit Yiew Instrument Automation Processing Help ball pje 2 S x Display Spectrum Profiles i Masses 1022 1080 I Press Ctrl and drag the mouse in Int 100 1429 mV mass 0 to 239387 x to this display selected display 100 will pan following the mouse 80 60 40 1000 1200 1400 1600 1800 2000 Mass Charge 100 0 m sum 3 m Profiles 701 750 Smooth Av 32 Baseline 10 4790 9 Selected display 4600 4700 4800 4900 6000 6100 6200 6300 5400 6500 Mass Charge For Help press F1 dM 135 13 M dM3 29 NUM Z Figure 20 48 Panning on a chromatogram display Panning displays 397 398 Chapter 20 Managing Data Displays Linking data displays Sometimes it is convenient to link the mass or shot ranges of data displays so that when the range of one display is changed another display is updated at the same time for example causing a list of peak masses to always show the masses of the 10 large
69. Green yellow Earth Green Maintaining compliance with the Electromagnetic Compatibility and Low Voltage Directives The following is a list of precautions which must be observed to maintain conformance with the European directives above Operate the instrument within the limits set out in either the technical specification or the site conditions sheet 1 Do not modify the instrument in any way electrically or mechanically 2 Ensure that all covers fan guards EMC gaskets and screws are fitted Ensure that all cable screens are connected Do not change component values Do not remove any labels Purchase spare parts from a recommended spare parts list Always replace parts like for like Keep a concise record of all the work carried out on the instrument and any parts changed with serial numbers where appropriate ON oO UB W Recommendation Arrange for an annual Portable Appliance Test PAT This test checks earth bonding mains loading and insulation integrity Warning Service work must be carried out by Kratos trained engineers Kratos Analytical cannot be held responsible for the action of untrained non Kratos service engineers who render the instrument non compliant with the above European directives Safety statements and warnings N Chapter 2 Safety warnings Safety labels Safety labels are fitted to the Axima instruments in a number of positions Please observe the warnings given
70. In the Criteria box Minimum intensity mV is the minimum intensity of the base peak Minimum S N is the minimum ratio of the base peak signal to the noise region Minimum resolution is the minimum acceptable base peak resolution Minimum S N specifies the fraction of the first accepted value s S N to which subsequent values may fall to before a new spot is selected and Maximum rejects sets the number of times a failing spot should be retried before a new one is selected The Auto aim box is used to set whether Prescan is applied the number of Profiles per point is the number of prescan profiles to be taken per raster point to be used in sorting The Minimum number of points is used as a minimum number of spots to be used should the Cutoff prove to be too severe in pruning the raster The Auto Calibration box is used to calibrate around a defined peak This attempts to compensate for inconsistencies across the individual sample Select the lock mass peak to calibrate around and the tolerance window around this peak The Auto Calibration will find the most intense peak within this window Interpolation is then performed assuming that a peak is found within the parameters The View Data button allows you to view and store data relating to an auto quality experiment Prescan Data Rank Intensity Count Sum Peak Intensity XY Position Well 1 769792 100 4 78 11 31 B1 2 769792 v 100 4 78 11 31 81 3 769792 v 100 4 7
71. It is not sensible to use auto quality when in MS MS mode and therefore in the Firing tab if you select the lon gate ON the Auto Quality feature is disabled The Auto Quality tab Figure 14 11 of the Acquisition tab dialogue is divided into four main box categories namely Monitor Auto power Criteria and Auto aim Acquisition default_linear Firing Exp Tech Auto Quality Storage Slide Raster Tuning Monitor Auto power Mass range EMi Power limits 30 60 Tt Start noise j Noise width 50 Start power 40 Criteria Auto aim Min intensity mY Min S N 5 4 Prescan V 1 Profiles Pt Min resolution Min S N 50 Min points 5 Max rejects Cutoff jo P Auto Quality Data J Prescan Data Il Overwrite Data View Data Auto calibration Lock Mass Calibration Lock Mass t4 Tolerance iz Performance measurement options C Abort experiment C Prompt for user s intervention Skip to next sample pey Figure 14 11 Auto Quality tab dialogue In the Monitor box the Mass range is that to be used for monitoring the base peak Start noise and Noise width parameters are used to identify a region to be used as typical of noise In the Auto power box Power limits and the start power are set Again cursor import can be used to set the Power limits range The start power should lie within the range Automated data quality filtering Chapter 14 Collecting data from a sample
72. LC MALDI Analysing Imaging Experiments Analysing Oligonucleotide Experiments Customer Support Guide Release Notes License Agreement About MALDI MS Figure 5 20 Help menu Help topics Open up an online help application which is a description of the menus and their functions User manual Opens up this user manual PDF within Acrobat Reader It provides a comprehensive manual of all the features Release notes Opens up a PDF version of the release notes supplied with the Axima within Acrobat Reader The notes describe various subjects that you need to made aware of when installing and using the software License agreement Opens a text file that describes the terms and conditions for using the software Help menu Chapter 5 Window and menu guides Graphical display sub windows angio2_msms0005 MALDI MS o x File Edit View Instrument Processing Help Sli e 2 5 X E Disper eri Shimadzu Biotech Kompact MALDI 6 v2 7 0 Build 20050524 Mode reflectron_ms_ms Power 73 Gate 1035 00 1060 00 P Ext 1050 bin 4 Profiles 1 54 Masses 176 1023 Data angio2_msms0005 4 Mar 2005 14 37 Cal tof 4 Mar 2005 14 37 CID of 1046 51 lnt 7 2 mV Profile 54 Click right mouse butto 100 fi has C Bek Pair ER Wa 6 6 m sum 356 mY Profiles 1 54 Unsmoothed Baseline 80 255 06 255 33 200 00 400 500 600 700 800 9300 1000 Mass Charge
73. Log Window an SoundMAX B MALDLMS JE Run f Startup i a a as Polymers Sae ei a a Sample Plate Editor 3 Log Off bill IT Volo View Express een i y z Turn Off Computer Figure 37 1 Starting the Element database The window shown in Figure 37 2 will be displayed Accessing the database Chapter 37 Element database Element Database BBE Atomic number Element name Element symbol Help Mass__ Abundance 1 00783 99 98500 2 01410 0 01500 l l l l l E elele le l le Figure 37 2 Element Database window The elements are listed in either Periodic table Atomic number Element name or Element Symbol order depending upon the tabbed property page selected For Atomic number Element name or Element Symbol order the element name elemental symbol average isotopic mass and most abundant isotopic mass are tabulated in a scrolling list Figure 37 3 Element Database BEBE i j Help Boiingpt l 1050 oo 3200 00 _Mass_ _ Abundance Ac Actinium 227 02780 227 EN i Ag Silver 7 107 86815 106 90509 2 961 93 2212 00 To ees Al Aluminium 13 26 98154 26 98154 1 660 37 2467 00 i i Am Americium 35 243 06140 243 06140 1 994 00 2607 00 Ar Argon 18 39 94768 39 96238 3 189 20 185 70 As Arsenic 33 74 92159 74 92159 1 817 00 613 00 At Astatine 85 209 98714 209 98714 1 302 00 337 00 Au Gold 79 196 96654 196 96654 1 1064 43 3080 00 B Boron 5 10 81103 11 00930 2 23
74. MS searches Chapter 18 Protein peptide analysis using Mascot search engine 5 Click the Open button the additional parent is added to the mass list Mass list search This section allows you to specify the list of masses on which to perform the You can add parent peaks to the search list where each parent entry contains a list of MS MS peaks The Add Parent button Fetches peaks from the currently loaded spectrum whereas Add File s allows you to fetch peaks from many data files at once You can then search Mascot directly using this list or save the list to a Generic Mascot file for use elsewhere Paroneand Parent 1722 958 Da ragment list for ID REBE RN GIT Eee MS MS search ithe Save List As Clear List Delete Peak Add File s Add Parent Save list as You can save the mass intensity peak list in a Generic Mascot Format mgf files This format allows you to use the list with third party software tools Searching the Mascot database 1 Click the Search button at the bottom of the window Cancel The Database search window is displayed while your PC connects to the Mascot search engine Database search xi A search is currently being performed The progress of the search can be seen below Search status Communicating with the search engine If there is problem accessing the search engine details are provided within this window Mascot MS MS searches 313
75. Mascot V Output gt Get Manual Settings Data Storage QIT MS MS Laser Firing Tuning mode positive Mass Range fico 10000 0 Calibration S Laser Firing Power po a seo oie 4 Profiles 10 per sample Shots Off 7 accumulated per profile Laser Rep Rate 10 0 H MS MS Resolution Std Isotopic Selection 250 7 CID Control I Restrain peak cleanup Save Method Load Method Figure 15 10 Method Editor QIT Laser Firing in MS mode For fragmentation of Precursor ions as a result of MS acquisition the parameters can be configured from the window shown in Figure 15 10 above Resolution Normally the 250 resolution window would be recommended as this will generally retain a complete isotopic distribution but will reject other close distributions e CID control Specifies the amplitude of the excitation waveform that is used in fragmenting the precursor ions A value of around 300 is recommended as a good starting point However some ions fragment more easily than others and it may be necessary to vary this parameter to obtain the best quality fragmentation Method Editor Chapter 15 Automated operation NB If this value is set to 0 then the correct precursor selection may be verified Calibration The Method Editor Calibration window displays similar functionality to the MALDI MS Calibration window described in Instrument Calibration on page 459 To acces
76. Mass Charge For Help press F1 Figure 17 5 Selecting the mass range using the mouse 274 Displaying Spectra Chapter 17 Viewing the collected data File Edit Yiew Instrument Automation Processing Help bel JE 2 a x x Display Spectrum Profiles fi Masses 1232 1305 l gt I Data pep_mix_2466d0005 110 c 10 Feb 2005 10 06 Cal tof 10 Feb 2005 9 02 Shimadzu Biotech Kompact MALDI 6 V2 7 0 Beta 1 Mode reflectron_ms Power 45 Blanked P Ext 2460 bin 9 Yolnt 1 5 m sum 220 mV Profiles 1 142 Smooth Av 20 Baseline 80 1297 18 100 90 80 70 i 1298 12 50 40 30 1294 1296 1298 1300 1302 1304 ie Mass Charge For Help press F1 Figure 17 6 Graph redrawn with new mass range Typing in an incorrect mass range or selection using the mouse can be undone by pressing the toolbar Undo button 2 This will set the Mass range to its previous value A printed copy of the displayed graphs can be obtained by pressing the toolbar Print button amp Automatic printing is available during data collection using the Display Options General options tab from the View menu Data collection will run much faster if the displays are updated infrequently Averaging a large number of profiles and updating after the average is much faster than updating the display after Displaying Spectra 275 276 Chapter 17 Viewing the collected data every profile It may be advantageous wit
77. Mass range 9000 12000 ti Method Gradient M 14 Signal Smoothing Average Ne Average Largest Segments 20 Combine Peak width 4 a Profiles Peak height 1 3 my Detect peaks Peaks detected Order Update Descending Ascending Profiles Mass Both Profile tagai Height Mass Profies Apex mV Profiles Masses 11250 11100 11100 1900 11100 Peak display Hee Previous First Last _ Gear 4 peak s selected Figure 30 2 Chromatography window Peaks within the selected mass range are detected if they meet the following conditions the peak height is above the value of the Peak height entry and the peak width covers Peak width profiles The values for Peak height and Peak width should be set as required to restrict the number of peaks detected to significant peaks within the collected data The selected mass range within which to search for peaks can be split into smaller sub ranges This assists in determining where the peaks maximise in the mass range Since chromatographic peak detection searches through profiles looking for regions of increased intensity these regions are located and recorded on the basis of their profile position not their mass position For this reason it is helpful to subdivide the mass Chromatographic peak detection Chapter 30 Chromatography range into smaller regions to locate peaks in terms of mass as well as profiles This is accomplishe
78. Multiple PrintType values are allowed Block lt MSx gt lt Output gt lt Export gt lt ExportDestination enumeration gt PC UNIX lt Columns unsigned integer gt gt 0 lt Delimiter enumeration gt comma space tab hash lt DecimalPlaces unsigned integer gt ASCII Text Method file format 221 Chapter 15 Automated operation lt ExportType enumeration gt headings profile average processed peaks lt Format enumeration gt intensity mass massintensity Multiple ExportType values are allowed 222 ASCII Text Method file format Chapter 15 Automated operation Displaying Auto Experiment Results Experiment results can be displayed within the MALDI MS application by opening a results file A results file is opened by selecting a file using the file open dialog box displayed when Open Auto Experiment Results is selected from application s File menu The display is a summary of what samples were analysed and what tests were carried out on those samples during an auto experiment The results take the form of a tree structure You can view the results of your experiment using the Auto Experiment Results viewer J MALDI MS of x File Edit View Instrument Automation Processing Help bel yael 2 8 Display Auto Experiment Results 7 Profiles 1 200 X Masses 1 700 14 gt C Documents and Settings roberto My Documents LC MALDI Results 6 protein mix 3 Resuits rex Well Sa
79. Points is the number of points in a regular raster 200 maximum e Type is either R for regular or F for freehand These are followed by the name of an input text file and the name of the raster file In the case of a regular raster i e tR the input filename is a dummy argument as all raster points are calculated The utility can also take a s Spacing argument as an alternative to p Points In this case the spacing between points in microns is specified If both p and s arguments are supplied the last entered is used in the calculation For a freehand raster the ASCII text file for input must contain a line of text for each point where each line contains the X and Y point coordinate in microns from the raster centre If tF is used then both p and s arguments are ignored and points are read from the input text file until the end of file is encountered As an example ascii2raster w5 0 h5 0 x0 0 y0 0 p100 tR dummyname txt reg100 rst will create a one hundred point regular raster which is a 5 0 microns square centred on the well to which it is attached Raster laser firing Chapter 13 Preparation for data collection Tuning for an acquisition Axima models The Tuning tab window is provided to allow advanced users only to fine tune the parameters which can be set for a particular acquisition Acquisition default_linear Firing Exp Tech Auto Quality Auto Sequence Storage Slide Raster Tuning Tu
80. RA Display Spectrum Profiles 1 Masses 12 1040 1 gt Figure 8 4 Frame bar displays the name of the loaded datasets Up to ten datasets can be loaded concurrently Loadingdata 78 Chapter 8 Loading and unloading data Unloading data Unloading data To unload any dataset which is being used by MALDI MS click on the unload button es adjacent to the dataset name to be unloaded The dataset will be unloaded and its name removed from the Load data window Alternatively to unload all datasets in one operation press the Unload all button Chapter 9 Parameter sets Chapter 9 Parameter sets 80 Chapter 9 Parameter sets Introduction Introduction The settings on each window in the MALDI MS program can be stored in what is called a parameter set By using parameter sets any number of users of the instrument can store and recall their own particular set of instrument parameters without affecting any other user Specific data collection and data processing settings can be stored in different parameter sets These can be reloaded later so that the instrument can be ready to collect data without manually adjusting large numbers of parameters All of the settings window positions displays and display types are also saved within the parameter set so that when operators load their specific parameter sets the windows appear in the positions in which they were saved Chapter 9 Parameter set
81. SELECT button on the number of the trace above the colour well in the Traces section The standard Windows colour palette will be displayed Figure 24 3 Changing spectrum trace colours 2431 432 Chapter 24 Choosing user defined colour schemes Color Basic colors El CS EE Z eee EER EE ERE Ee EEE Ee EEE Ee Custom colors E pm Hue 160 Bed 255 safo Green 255 ColorlSolid Lum 240 Blye 255 Add to Custom Colors Figure 24 3 Windows colour palette On this window the colour can be defined either by using the mouse and selecting from the colour palette or by selecting the RGB Red Green Blue components of the colour Click on OK to accept the selection This process can be repeated for all 10 charged and neutral traces until the required colour combinations have been defined Other items which can be defined are colours for Unresolved peaks Significant peaks manually Mass assigned peaks the Subtracted baseline and Peak limits markers Centroided peaks are displayed in the trace colour for each dataset however there are three cases in which it may be favourable to distinguish certain peaks from the others In the case of Unresolved peaks peaks which do not reach the user specified baseline threshold it would be better to know that a peak is unresolved rather than leaving the peak unlabelled Significant peaks are peaks which have been characterised as belong to a specific group e g in
82. Securit 5 canes m ul e se caren eres Sample Plate Editor olo View Express on g2 Search Figure 3 3 Starting MALDI MS from the taskbar The MALDI MS program will appear on the desktop Introduction 27 28 Chapter 3 Getting started Introduction to the MALDI MS software When the MALDI MS program starts the instrument will begin its initialisation routine and the main control window will appear on the screen Figure 3 4 J angio2_msms0005 MALDI MS iof x File Edit View Instrument Processing Help Bla Hee 2 BN Ol Dissectum Profies 1 54 Masses 1761023 LL Data angio2_msms0005 4 Mar 2005 14 37 Cal tof 4 Mar 2005 14 37 CID of 1046 51 Bz Shimadzu Biotech Kompact MALDI 6 v2 7 0 Build 20050524 Mode reflectron_ms_ms Power 73 Gate 1035 00 1060 00 P Ext 1050 bin 4 lnt 7 2 mV Profile 54 100 6 6 m sum 356 m Profiles 1 54 Unsmoothed Baseline 80 255 08 600 Mass Charge For Help press F1 Figure 3 4 The MALDI MS base window This window will be referred to as the base window since this is the window from which all other menus and sub windows are invoked As long as the base window or its icon is visible on the taskbar then the MALDI MS program is active and available for use The instrument can only be controlled by one MALDI MS program This is to avoid conflicting commands or incompatible operations being sent to the instrument by different programs Intr
83. Singly charged N Terminus C Terminus jazze a b bet bea p17 oae ve ya v yaz vasl z e2 c1 c z122 ajejifal vaje Clear Charge Multiply charged m z range 7 500 Max charge f5 a ok Cancel eptide Settings Keyboard Display Fiagmertation Match Peaks Nested PSD Specta Display Font size MEYE Colouman Colour Re Keyboard Short symbol Y Sequence Short symbol Y Report Include linked sequences V Report mass 2 Decimals OK Cancel Keyboard Display Fragmentation Match Peaks Nested PSD Spectra Select peaks Single sequence peak match Report missed experiment peaks V Search Currently loaded sequence Report missed theoretical peaks IV o General Tolerance 2 afo E Match Ready ok Cancel Figure 5 18 Peptide Settings tab options Settings a Processing menu Chapter 5 Window and menu guides i Peptide Settings g Peptide Settings Figure 5 19 Peptide Settings tab options Settings b Processing menu 54 Chapter 5 Window and menu guides Help menu This menu provides access to useful information about the product See also page 34 Help Topics User Manual Axima Performance Getting Started Guide Axima Confidence Getting Started Guide Axima Assurance Getting Started Guide Analysing Polymers Analysing Proteins using
84. Target plate user guide details all the available Target plates and how to use them This booklet is supplied with the Axima Application guides There are a series of application guides that describe how to use specific chargeable features These guides are supplied with these features Printed guides Chapter 1 Introduction Getting help eee On line help On line help is not available if you are using Windows Vista The online help provides procedural and reference information You can access on line help using any of the following from the Launchpad window click the Help button Launchpad PE E3 Info Show on startup V ee MALDI MS Search Archiver Enzymes Sakae Elements Compounds Polymers press the F1 key the on line help opens at the relevant topic click the button and then click the area of interest the on line help opens at the relevant topic e click the Help button on the top toolbar Tool tips Tool tips provide extra information when you move the mouse pointer over a field for example Shots 2 X bi accumulated per profile Number of laser shots to accumulate per profile Getting help Chapter 2 Safety warnings Chapter 2 Safety warnings Chapter 2 Safety warnings Health and safety precautions Warnings and cautions For your safety and the safe operation of the Axima read the following warnings and cautions Warnings highlight situations th
85. URL for a local Mascot Server is http lt server name gt mascot cgi nph mascot exe 1 Enter the name of the server where the FTP results are to be stored Enter the name of the ftp results location commonly setup at as mascot_data This is the FTP results site defined on the Mascot Server as the path to the Mascot data directory commonly C I netpub Mascot data Enter the name of the FTP intermediate processing site This is the path to the newly created directory on the Mascot Server commonly C lnetPub Mascot data temp_results Enter the name of the FTP configuration location This is the FTP config site as defined on the Mascot Server commonly C lnetpub Mascot config Set this tick box to protect any FTP file transfers between the Mascot Server and the Axima PC by a Username and Password otherwise Anonymous login is assumed Set the User Name to use for FTP connections Set the Password to use for FTP connections Chapter 6 Configuring Launchpad After making any changes click the Apply button Mascot Setup 65 66 Chapter 6 Configuring Launchpad Mascot Setup Chapter 7 The Log Window Chapter 7 The Log Window Chapter 7 The Log Window m Windows event viewer The Axima software takes advantage of the Windows Event Viewer which is part of the operating system To open the Event Viewer 1 From the Launchpad window click the Log icon Launchpad
86. Use this option to perform PMF Peptide Mass Fingerprint or MS searches Internet search Internet search amp Select the engine to be used for your search From the list below Search engine Mascot PMF 1 In the Search engine field select Mascot PMF from the drop down menu Search settings Search settings Z This section allows you to specify a webpage URL to be used when submitting searches Once selected you can choose the type of database you wish to use Search URL g http www matrixscience com cgi nph mascot exe 1 Database Icon is displayed while MALDI MS attempts to connect to the server 1 The Search URL field is usually displayed automatically The Matrix Science database URL is http www matrixscience com cgi nph mascot exe 1 296 Mascot PMF searches Chapter 18 Protein peptide analysis using Mascot search engine If you are using a local Mascot server the URL is typically http lt mascot server name gt mascot cgi nph mascot exe 1 If you experience problems accessing either the I gt Internet or your Mascot server Ask your IT department to check the access rights from the PC Check that the correct parameters have been set up in the Environment Configuration Editor default path to the editor is C Program Files Shimadzu Biotech Launchpad Programs config_environment exe If you change the URL the Refresh button is displayed Click it to con
87. V5 Gate valve Vacuum state Status Open Closed Open Closed Open Closed Start System Vented Analyser Pumping Roughing Source Source Pumping System Pumped Explanation The valve is shown Open when the SAC turbo pump 1 is vented This manual valve is shown Open when the flight tube turbo pump 2 is vented Isolates flight tube from analyser for door opening Shown as open when instrument is acquiring Instrument just switched on System is vented at atmosphere for maintenance Pumping down Flight tube Begin to pump down the source Pumping down SAC only Instrument is pumping ready for data collection Note The vacuum system shows System pumped when this pumping starts The system will not be ready to collect data until one minute after the turbo pump is up to speed and the vacuum gauge reads 2x10 Axima Confidence instrument status 99 Chapter 11 Checking the instrument status Axima Performance instrument status The instrument status diagram gives an overview of the instrument status in real time Instrument Status Vacuum state Gauge 2 System pumped Tuning mode reflectron_ms rob Instrument mode Reflectron CID Gas V4 Purge Vi e Gauge 4 Gauge 1 4 3E 6 mbar Door closed Pump Supplies Pressure Valves Pulsed Source Extraction Lens X motor Atspeed On OK Open A Source Source Linear d off
88. Vann wane wy 545 550 555 560 565 180 185 190 195 200 MasgCharge MasgChamge Axima Performance Chapter 28 Fragment ion calibration m Axima Confidence The aim of the fragment calibration procedure is to define as accurately as possible the relationship between the apparent mass and the actual fragment mass Moreover this must be done for the mono isotopic masses peaks only the isotope peaks corresponding to no C carbon or only C isotopes in the ion the lowest mass isotope in the distribution Apparent mass Actual mass Fragment varies between mono isotopic peak instruments only y14 1469 1436 81 y13 1405 1339 75 y12 1242 71 y11 1145 65 y10 1048 59 y9 951 54 y8 854 59 y7 757 44 y6 660 38 a6 555 33 a5 458 28 a4 361 22 b2 521 195 11 b1 422 98 06 Table 28 4 Fragment calibration peaks for P14R Axima Confidence The table above contains a list of the fragment peaks for P14R The name of the fragment is in the first column and its correct mono isotopic mass Actual mass is in the third The middle Axima Confidence 489 490 Chapter 28 Fragment ion calibration column Apparent mass contains the approximate mass at which the fragment peak appears when the fragment calibration is not applied i e when the normal calibration is active Data p14r_msms0004 13 c 28 Sep 2005 13 35 Cal fe_test 28 Sep 2005 13 34 CID of 1533 66 Data p14r_msms000413 c 28 Sep 2005 13 Shimadzu Biotech Axima T
89. Warning 12010 Some Compound definitions used by the sequence calculator may be missing see log window for details The loaded sequence may include units not defined in the current Compound database on this computer see the Window event log for the specific unknown units Warning messages 661 662 Chapter 44 Summary of error messages Warning 18000 The compounds database may be an old version please update This is not a current version of the Compounds database it should be updated to a new version using a later release of MALDI MS software Warning messages Chapter 45 Bibliography Chapter 45 Bibliography 663 Chapter 45 Bibliography 664 T Ito G J Q Van der Peyl P G Kistemaker and J Haverkamp Laser lonization Mass Spectrometry Mass Spectometry Vol 30 No 3 205217 1982 H J Heinen S Meier H Vogt and R Wechsung Laser Desorption Mass Spectrometry with LAMMA Fresenius Z Anal Chem 308 290296 1981 H Heinen On lon Formation in Laser Desorption Mass Spectrometry with LAMMA Int J Mass Spectrom lon Phys 38 309322 1981 R J Cotter Mass Spectrometry of Nonvolatile Compounds Desorption from Extended Probes Anal Chem Vol 52 No 14 1589A 1604A 1980 K Tanaka H Waki Y Ido S Akita Y Yoshida and T Yoshida Protein and Polymer Analyses up to m z 100 000 by Laser lonization Time of Flight Mass Spectrometry Rapid Communications in Mass Spectrom
90. a polymer series peaks belonging to a specific polymer series will be flagged as significant and these peaks can be drawn in a different colour to distinguish then as part of a polymer series Manually Mass assigned peaks are peaks to which the user has manually assigned a peak mass as opposed to the program automatically assigning peak masses The subtracted Baseline can be drawn on an averaged data trace to indicate the amount of baseline noise which was removed This line can be drawn in a specific colour Changing spectrum trace colours Chapter 24 Choosing user defined colour schemes The position of the Peak limits can be indicated to show the area of the peak which is being used to calculate the peak centroids Having made all of the required selections press the Apply button to apply the selections to all of the displays This will apply the new colour scheme to the displays but the colour scheme modifications will not be saved in any way they are only temporary To save the user defined colour scheme so that it will be made the default colour scheme and loaded automatically when the MALDI MS software is loaded next time press Save If the colours have been modified for a temporary change and the previous colour scheme is required to be reinstated press Load and the previously saved scheme will be loaded Press Load defaults to use the factory defined MALDI MS colour scheme Changing spectrum trace colours 433
91. and the nearest higher loaded but deselected dataset is selected in its place The window is now set up to display all four types of spectrum traces Experiment with the settings on the Spectrum Display window to familiarise yourself with the options on the window and their effects upon the displayed data traces Multiple sample datasets Chapter 12 Introduction to displaying data Usually the Profile and Averaged traces are of most interest for people wishing to see data as it is being collected The Processed trace is preferred to see the results of data processing such as smoothing baseline subtraction peak centroiding etc after data has been collected These various options will be discussed in detail in later sections Particularly for the Axima instrument family it is useful to be able to display more than one sample from a dataset To do this 1 Select the Display multiple samples check box 2 Enter the samples to be viewed in the appropriate Multi sample edit box or alternatively click on the plate button to the right of the Multi sample selection to invokes a popup display of the plate with acquired samples indicated All acquired sample can be selected or deselected using the buttons at the bottom of the window or individual samples can be selected using the mouse 3 Select OK to automatically enter the selected samples in the multi sample edit box Select acquired samples Figure 12 5 Axima multi sample sel
92. and one Glycine molecule from the Combined_ang_brad species to produce a new entry Where an elemental group is being subtracted e g CH3 COOH etc this should be placed within parenthesis otherwise only the first element is subtracted i e C in CH3 For example Formula Acetic_acid COOH 4 Acetic_acid COOH 8 If an invalid name or formula is typed in then an error will be reported More detailed information on the nature of the error will be found in the error messages in the Console window Where a general compound definition is used in another compound definition for a database entry the general compound must be placed within angled brackets lt gt to avoid confusion with another definition which may have the same name i e where Phe could be phenylaniline or phenylalanine lt Phe gt unambiguously denotes Phe to be a general compound definition rather than the amino acid definition Chapter 38 Creating a compound database Sorting and showing compound definitions Using the Sort option entries can be sorted in alphabetic order category order or mass order simply select the option required The list can also restrict the display to specific compound by setting Category to the specific category required Figure 38 10 shows a list sorted in alphabetic order and showing only the Sugar category definitions Compound Database Bek Category Sua M Sort Alphabetic New Delete Export Help Compound Formul
93. and release the mouse button The selected display will be copied to the new location Figure 20 24 Copy insert and delete displays 365 Chapter 20 Managing Data Displays p14r_msms0004 angio2_msms0006 MALDI MS of x File Edit View Instrument Automation Processing Help sal g je 2 al x a Display Spectum T Profiles fi Masses 515 531 rel lnt lnt 3 a202 Still holding the SELECT 100 button down drag the setetted s display to its new destination f se 4 and release the mouse button 40 40 20 0 o 500 1000 1 c f 550 Mass Charge Mass Charge lnt a 2ic pint Tfc F1 522 03 n 451 94 49209 19 05 0 17 2c 450 500 550 1 e Fi MassiCharge For Help press F1 1 Select the display to copy 2 Click on the iq button 3 Using the left mouse button click and drag the selected display to its destination ff p14r_msms0004 angio2_msms0006 MALDI MS of x File Edit View Instrument Automation Processing Help JEES 2j x a Display Spectrum Profiles fi Masses 515 531 Mass Charge Mass Charge 5p2 03 P 451 94 49209 19 05 0 2c 450 500 550 1 c F1 520 525 Mass Charge Mass Charge For Help press F1 ldm 68 34 MjdM 7 40 4 Figure 20 23 Copying a display 366 Copy insert and delete displays Chapter 20 Managing Data Displays p14r_msms0004 angio2_msms0006 MALDI MS o File Edit Yiew Instrume
94. angio2_ Figure 26 5 Display Contents for Instrument Record Listing 458 Summary of run wide conditions Chapter 27 Instrument Calibration Chapter 27 Instrument Calibration 459 460 Chapter 27 Instrument Calibration F p14r_msms0004 MALDI MS olx File Edit View Instrument Automation Processing Help zli Hle 4 al x Calibration Calibrant references m Calibration files Dataset None Cursor mass l Insert Delete Tolerance a00 mDa z 2 Mass Ure Distabution Resolution r R HaT H Formula Calculate average x l Calibrate Undo Combined Cal Introduction Before analysing a sample for molecular weight information the Axima instrument must be calibrated with a suitable calibrant compound or mixed calibrants covering the mass range of interest This calibrant sample is applied to the sample stage as with any other sample and a number of shots at the sample are made to obtain a characteristic spectrum The calibration window provides all of the tools needed to create reference files and calibrate the instrument within one window To use the calibration window select Calibration from the Processing menu the Calibration window will appear Figure 27 1 Annotation Peak Processing Chromatography Polymers Sequence Calculator Reference Editor Select Peaks Manual Peak Assignment BBE a Reference ed
95. at the position at which the laser will fire As indicated in Figure 14 13 holding the right mouse button down over the camera image produces a menu with six options these are as follows e Live Freeze Initially the camera image is live i e continuously updated and the Live option of the toggle pair is disabled Selecting Freeze stops updating of the image and disables the Freeze option until the now available Live option is selected Copy mage places a bitmap copy of the camera image on to the clipboard e Save Image invokes a standard file dialogue window which allows the camera image to be saved to file Settings invokes the popup menu shown in Figure 14 13 which controls the camera image The usual image controls are available for example slider bars are used to control brightness contrast hue and saturation The image can be digitally zoomed automatically fitting the image frame by use of the zoom slider bar Check boxes Horizontal and Vertical Mirror may be used to cause the image to be reflected in the respective mirror planes The Cross hairs and 1mm Scale both shown on the image in Figure 14 13 can be toggled on or off using the check boxes Alignment marks can be enabled causing an oval image of the aperture to be shown which is used to align the image so that the cross hairs are centred at the laser focus The set of four radio buttons BW CCIR BW RS170 PAL and NTSC is used to identify the type
96. available when data have been stored using the Store profiles All or After average option On the Peak clean up window the Average option specifies whether All profiles in the range displayed or only the Tagged profiles are averaged By selecting Tagged profiles only profiles tagged as described are added together This generally gives better results than if profiles were included with little or no information in them Combining tagged profiles Scenario Chapter 16 Cleaning up data Select the scenario appropriate to your experiment only the parameter fields appropriate for you setting are displayed Selecting the Advanced scenario displays all the fields Option Scenario options Description Non isotopically Defaults to using resolved peaks I sotopically resolved peaks Peak harvester Advanced Average smoothing Threshold apex peak detection algorithm Defaults to using e Gaussian smoothing Centroid based peak detection algorithms Threshold 25 centroid detection algorithm This morphological peak detection algorithm has no user settings Subsequently Poisson modelling is applied to determine which peak in an isotopically resolved group represents the monoisotopic mass of a peptide References a P Soille Morphological Image Analysis Principles and Applications Springer Verlag Berlin 1999 b Breen EJ et al Automatic poisson peak harvesting for high
97. be automatically updated This could be used for example to compare the data from two continuous slides by making two chromatogram displays with a link between them 400 Linking data displays Chapter 20 Managing Data Displays Amplification Regions of low signal strength on a graph can be amplified to show finer detail which may not normally be visible The region to amplify is marked by the range cursors the cursors must be on the selected display The display toolbar amplification buttons provide the amplification factors shown in the table below Table 20 8 Amplification factors available from the toolbar buttons 5 Shift amp normal Shift Control Ctrl x1 Cancel all x100 oo amplification x2 x20 x200 x2000 we x5 x50 x500 x5000 gS x10 x100 x1000 x10000 nao Pressing 1 2 5 Or i0 amplifies data by the factors shown on the Controibar button a combination of the keyboard Shift and Ctrl keys with these buttons give the other factors shown in the table Pressing Shift and a cancels all amplification regions marked in the selected display and returns the signal back to its original value Figure 20 50 shows an example of a display using amplified regions Amplification 401 402 Chapter 20 Managing Data Displays 10x amplification 5x amplification angio2_msms0006 pep_mix_2466d0005 p14r_msms0004 MALDI MS File Edit view Instrument Automation Processing Help ia ee 2
98. be labelled with one of the sequences by first highlighting the sequence in the candidate list then using the right mouse button to bring up the menu over the list and selecting the label option from this menu Matching spectral peaks with theoretical fragments Introduction A useful feature in any data system is the ability to compare spectral peaks from collected data with libraries of spectral peaks in an attempt to match peaks within the spectrum The Sequence Calculator allows peaks within the collected data to be selected and compared with fragment databases Chapter 42 Sequence Calculator First select a display within the MALDI MS base window showing a spectrum of collected data and choose Select peaks from the Processing menu The displayed window is shown in Figure 42 31 Select Peaks x Dataset 1 angiotensinogen_digest_005 Automatic Masses 50 10000 el Peaks 30 E J Manual Mass eg Edit Mass List Insert Delete gt All masses v Figure 42 31 Select Peaks window Two modes of peak selection are available peaks may be selected either automatically or manually The automatic selection chooses the most intense peaks in a given mass range For manual selection masses of individual peaks may be typed or indicated using the cursor When selecting peaks avoid peaks at low mass e g below mass 300 as low mass fragments often occur at the same masses in digestions of pept
99. calibration applied In the Exp Tech window set Reflectron mode e Mass range 0 8 000Da Laser rep rate 10Hz In the Firing window set 10 shots per profile 50 200 profiles Blanking 300Da e Pulsed Extraction 1534Da Collect a normal ms spectrum from a P14R sample Use gausssian smoothing of 20 and baseline subtraction 80 Calibrate on the P14R parent monoisotopic peak at 1533 86Da and the Cyano2H peak at 379 09Da Save the calibration With the ion gate set to 1520Da to 1550Da collect 10 ten P14R fragment spectra settings as above all with the parent mass within lt 0 1Da of the correct value of 1533 86Da Aim to achieve good quality fragment peaks in all of the fragment spectra Load all ten P14R fragment calibration spectra into the Maldi_MS data sets Set the processing to data set number 1 in the traces window Inthe calibration window switch off the fragment calibration by selecting remove in the fragment calibration mass section mid right hand side of the window This will apply the normal calibration to the fragment spectrum the fragment calibration is switched on automatically after collecting a spectrum with the ion gate on Axima Confidence 2491 Chapter 28 Fragment ion calibration 9 The fragment calibration setup window is accessed from the calibration window by selecting set up in the fragment calibration mass section mid right hand side of the window H
100. corner of the window footer bar Figure 20 13 J pep_mix_2466d0005 MALDI MS olx File Edit Yiew Instrument Automation Processing Help bel ay 2 a h lt Display Spectrum Profiles fi Masses 1040 1058 Ie Data pep_mix_2466d0005 11 Ofc 10 Feb 2005 10 06 Cal tof 10 Feb 2005 9 02 Shimadzu Biotech Kompact MALDI 6 2 7 0 Beta 1 Mode reflectron_ms Power 45 Blanked P Ext 2460 bin 91 lnt 0 5 m sum 69 mY Profiles 1 142 Smooth Av 20 Baseline 80 1047 46 Cursors OFF button Range between the cursors is shown here 1042 1046 1048 1050 1052 4 1056 1058 e Mass Charge Mass 1049 79 Closest peak M dM 2287 29 dM 3 94 MjdM 266 11 T Figure 20 13 Both range cursors on a display Repeat step 1 and 2 to obtain a second vertical cursor The mass difference between the two cursors is displayed in the footer bar at the bottom of the graph as dM A measure of the resolution between the cursors is also displayed as M dM where M is the centre mass between the two vertical cursors and dM is the mass difference Cursors 353 Chapter 20 Managing Data Displays When both vertical cursors are displayed on the screen pressing and holding down the mouse ADJ UST button causes the nearest vertical cursor to the current mouse position to jump to the mouse position This allows rapid repositioning of the cursors Cursors are erased by using the cursors off button from the graphical displa
101. criteria Ro Enter any known details about the sample and its preparation Taxonomy Arabidopsis thaliana thale cress Digest enzyme Trypsin Missed cleavages Fixed modifications Acetyl K Acetyl N term Acetyl Protein N term Amidated C term Amidated Protein C term Ammonia loss N term C Biotin K Biotin N term Variable modifications Acetyl K Acetyl N term Acetyl Protein N term Amidated C term Amidated Protein C term Ammonia loss N term C Biotin K Biotin N term zl Protein mass 0 kDa Treat masses as Monoisotopic C Average Peptide tolerance 0 3 Peptide tolerance unit Da w Mass Type m x I Decoy In the Taxonomy field select the required source of the protein In the Digest enzyme field select the required enzyme and in the Missed cleavages field select the tolerance From the Fixed modifications and Variable modifications fields if required select the appropriate modifications You can select more than one modification For the Protein mass field generally do not use this field as it may hinder the search This field is in kilo Daltons For Treat masses as click the appropriate radio button In the Peptide tolerance field enter the required tolerance and in the Peptide tolerance unit field select either Da or mmu millimass unit from the drop down list In the Mass Type field select the required type from the drop down list For an automatic dec
102. data collection Acquisition positive Firing Exp Tech Auto Quality Storage Slide Raster Tuning OlT ToF MS Tuning file Save Voltages Intro Endcap M Reflectron Centre 4 4 Y lon Polarity iy Extr Endcap Zi Y Reflectron Back Yy ToF Float Y Detector Yy Print Adjust Figure 13 3 Axima Resonance Analyser tuning window parameters Setting up MS parameters in the Axima Resonance The Axima Resonance has an extra QIT ToF MS parameter tab Figure 13 4 below which sets the parameters for MS to MS experiments 4 Acquisition positive Firing Exp Tech Auto Quality Storage Slide Raster Tuning QIT ToF MS MS MS Precursor Ion 1046 540 laa Precursor Ion 0 t Resolution Wide Isotope Selection 70 X Resolution Standard Resolution d CID Control CID Control Adjust Gate Clear All Apply Tuning for an acquisition 137 138 Chapter 13 Preparation for data collection Figure 13 4 Axima QIT ToF MS window parameters At the top of the window the arrows to the right and left allow the parameters for specific stages in the MS experiment to be adjusted These parameters are as follows Precursor ion the ion selected for fragmentation which should be progressively smaller Note however that at each stage the ion trap will retain ions approximately from the precursor down to 1 4 of the precursor The precursor ion may also be s
103. directory being written to Error messages 651 652 Chapter 44 Summary of error messages Error 9020 Unable to create new data folder Retry the operation check directory write permissions on the directory being written to and disk space Error 9030 Cannot open the filetype file filename Check that the file filename exists in the selected directory Error 9040 Cannot write run_ stats to the run file Retry after checking file and directory write permissions on the directory being written to and disk space Error 9050 Error reading Run_ stats from the run file The Windows event log may contain more specific descriptions of the errors encountered check the size of the run file ensuring it is not zero Error 9060 Error reading the named file The Windows event log may contain more specific descriptions of the errors encountered check the size of the run file ensuring it is not zero Error 9070 Error writing the slide number to the named file Error 9080 Error writing raw data header Error 9090 Error writing raw data Error 9100 Error writing raw data profile terminator For all of the above retry the operation after checking file and directory write permissions on the directory being written to and disk space Error 9110 Memory not available for creation of output buffer Close other programs shutdown active processes to free more memory Error 10000 Could not allocate timer to drive the system The application will t
104. e 2 S neki Profiles 1 Masses 12 1040 1 gt I Peak Processing Chromatography ce Calculator Reference Editor Select Peaks Manual Peak Assignment Internet Search Figure 42 1 Starting the Sequence Calculator The Sequence Calculator window will be displayed Figure 42 2 Sequence Calculator Biel Es File Edit View Settings I gt gt gt Synchiorse Name United Resclution 1 penea z l i i e000 o g N terminus Hydrogen H Monoisotopic he C terminus Hydroxy H 0 x Fragmentation Singly charged z Generate Peak Markers Calculate Cation Proton H x Digest Length 0 Figure 42 2 Sequence Calculator window Introduction Chapter 42 Sequence Calculator The Sequence Calculator provides the following features Allows the creation of peptide and protein databases which can be searched for matches with a currently loaded sequence or spectral peaks Remote database searching via email or the internet is also available Prediction of fragmentation pathways for singly and multiply charged species Detailed analysis of theoretically predicted products from enzymatic digest and mass spectral fragmentation of protein and peptide chains This information can also be used in the calculation of theoretical distributions to simulate peptide spectra Calculation of useful peptide parameters such as hydrophobicity or hydrophilicity a
105. e g the wrong fragment isotope was found the reflectron is faulty HV breakdown or a faulty resistor calibration is acceptable then store the calibration to tof and close the fragment calibration setup Axima Confidence 493 494 Chapter 28 Fragment ion calibration Examples of peak shapes The examples below represent the various different shapes of peaks that you can expect from ms ms fragmentation 1534 03 1339 75 100 100 f 1535 80 344 07 90 90 J 20 U V oo 70 70 ate 1436 71 60 so 50 1 aR 39 40 40 30 20 20 20 s 1699 36 1549 S 0 y A 1531 07 s E 5 10 AL AZ ee ee 0 2 DA 1530 1532 1534 1536 1538 1540 1220 1225 1340 1345 1250 MasyCharge Masscnarge 1145 65 951 54 100 100 90 L a7 bad 70 70 7 60 50 40 40 j 20 30 20 20 N 10 94468 oj eee a Soy oj gt E N en Ae 1135 1140 1945 1150 1155 oa 015 20 255 MasgChamge MassCharge 46 03 195 11 100 100 555 33 90 90 80 80 70 563 14 70 60 60 50 50 f 40 40 20 30 20 20 ii 10 541 56 n j N i 10 N A j TER A o V VW AS Was of Vann wane wy 545 550 555 560 565 180 185 190 195 200 MasgCharge MasgChamge Axima Confidence Chapter 29 lon gate calibration Chapter 29 lon gate calibration 495 Chapter 29 lon gate calibration For Axima Resonance models only Introduction The ion gate filters out unwanted ions and only allows the required
106. ened 303 Other database SearcheS ccceeeee cece cence teeta eee eee tae teens 315 lon finder errr erence errr er eter rere re rr errr tr erie tere cere rere 317 I MEFOGUCHON raidsc ise E A A E AST 318 Importing a list Of MASSES cece eee eee eee eed 320 Creating editing a list Of MASSES 0 cece cece eect e eee eee eee 324 Defining destination of results file eeens 326 Saving and loading lon Finder sSettingS eceeeee eee ee es 329 Processing the data ccceceee cece e eee ee eee teeta ete eet ee teen a tees 331 Managing Data DisplayS c sccceeeeeeeeeceeeeeeeeeeeeeeeeeeueeeeuseeanenees 333 LMEPOCUCHION i onc cecc nce scenes veinna n e cela teeeeeweeecetens 334 The Display Toolbar c cc ceeeeee cnet e eect eee tee etna ne ed 337 Multiple displays ccccce eee e eee erence eee eee estate eae e es 342 Scrolling graphical displayS c cece eee eect eee ee eee eee e eee ed 349 CUIRS OMS naialiteidtutavaieadadaeasvathainy ARAA ene sarin EA TAARNA E a eens 352 Copy insert and delete displays ccceeeeee scene eee eae teens 365 Customising Graphical Reports 0cecceceee eee eeeee teeta eeaenes 371 Customising Text Reports ccceceee eee eee cnet e eats eee eaeee ene 380 Annotation osiers ccc eee ene ee eee aA enai aa aE maa 382 PANNING CiSPlAYS wi ccc oeceseee cree tirer n eevee ent ene 395 Linking data displays c
107. file In the Import field separator area select the radio button for the field separator used within the text file This field also defines the field separator for the results file produced when you select the Process button Inthe Reported peaks area select the required radio button to define which peaks to use when reporting intensities In the Required decimal places field define the number of decimal places for the results file Select the OK button Importing data from a file 1 Set the lon Finder settings window to interpret the data that you are going to import Importing a list of masses 321 Chapter 19 lon finder 2 In the lon Finder window select the Import button Open 7 x Look in e mass lists e My Recent Documents E Desktop 2 My Documents Fr My Computer Ka enna File name mass list txt z Open Places Files of type fal Files z Cancel Figure 19 4 Opening a text file 3 Navigate and highlight the required text file 4 Select the Open button glon Finder Ble ke m Ion search template CEE adden recne rt A Remove entries Remove all Results file destination Folder Folder file Filename I Auto select filename Dialog data Load new Save Process Figure 19 5 lon Finder window showing data If mass tolerance data already exists in the lon Finder window new data
108. for data collection A sample plate has three alignment reference points these will be the positions that will be used to correlate plate locations to stage locations The three reference points are usually the locations of three wells e g Al A24 and P24 The reference points are modified within the Plate Alignment References dialogue box which is opened by pressing the Alignment Refs button The co ordinates for the reference points are in the plate co ordinate system A reference point is modified by double mouse clicking on the required cell and modify it as required press the Return or Enter key to confirm entry A well location can be used by entering the well identifier into a Ref ID cell pressing Return or Enter then pressing the Get Well Location button see Figure 13 10 Settings are accepted when the OK button is pressed The plate file must be saved so that the changes can be accepted Plate Alignment References 21x References a Ref Ret iD Plate fmm Plate Y mm 1 Al 72 250 114 250 2 A24 72250 10750 Get well location 3 P24 4 750 10 750 Cancel Figure 13 10 Sample plate alignment references Creating plate files using the ascii2plate utility Alternatively a utility program ascii2plate is supplied with the Launchpad software This converts ASCII text input into a plt file The simplest way to use this utility is to first use a text editor to create an ASCII input file called for
109. icon To reverse the process simply select the required files and press the Decompress button Chapter 33 Exporting data and data displays Chapter 33 Exporting data and data displays 523 Chapter 33 Exporting data and data displays Exporting ASCII data There is often the requirement within laboratory environments to be able to perform statistical analyses upon experimental data This can take the form of testing the reproducibility of experimental data and the like For this reason the facility has been provided to export the collected data held within the MALDI MS window data buffers as ASCII data This data can then be imported into various spreadsheets for statistical analyses to be performed To export the data first display the sample profiles and mass range of the data which you wish to export in the normal manner The data which will be exported is the data shown within the chosen mass range in the currently selected window From the base window File menu select Export and from the sub menu select ASCII Figure 33 1 F p14r_msms0004 MALDI MS Oy x File Edit View Instrument Automation Processing Help Open Ctrl 0 Display Spectrum X Profiles 1 X Masses 12 1040 1 gt Git Save As Open Auto Experiment Results Comments Parameters gt Print Ctrl P Print Repor Print Preview Print Setup Window as a MetaFile Be Selected Tile as a MetaFile
110. if you wish to use this feature ICAT Isotope Coded Affinity Tag a method for protein quantification Mascot MS MS searches 307 308 Chapter 18 Protein peptide analysis using Mascot search engine Mass list 2 Set the four fields that define which fragment peaks are selected See the following diagram and explanation of these fields Mascot MS MS searches Chapter 18 Protein peptide analysis using Mascot search engine Parent mass Segments ae EO Low peak High peak percentage limit percentage limit The Low peak percentage limit and High peak percentage limit fields set the boundaries for selecting peaks for the fragment mass list The limits are set as a percentage of the parent mass Peaks outside the boundary are ignored The Max number of peaks and Segments fields define the maximum number of peaks and their distribution For example if you set the maximum number of peaks to 40 and the number of segments to 5 the software e divides the spectrum in to 5 equal segments e from each segment picks the 8 40 divided by 5 most intense peaks uses the 40 peaks for the fragment mass list Mascot MS MS searches Chapter 18 Protein peptide analysis using Mascot search engine 3 Click the Fetch Peaks button the fields are populated with the fragmentation data obtained from your spectrum Add MS MS Mass List Ea Use this window to select MS MS peaks from the curre
111. in Normal and Tolerance cursor modes The numbers 2 to 10 i e Tolerance and Additional mass and charge cursors specify the colours of the multiply charged or multiple mass cursors which track the movement of the Normal cursors see Cursors on page 352 Having made all of the required selections press the Apply button to apply the selections to all of the displays This will apply the new colour scheme to the displays but the colour scheme modifications will not be saved in any way they are only temporary To save the user defined colour scheme so that it will be made the default colour scheme and loaded automatically when the MALDI MS software is loaded next time press Save If the colours have been modified for a temporary change and the previous colour scheme is required to be reinstated press Load and the previously saved scheme will be loaded Press Load defaults to use the factory defined MALDI MS colour scheme Changing cursor colours 439 440 Chapter 24 Choosing user defined colour schemes Changing sequence calculator colours The sequence calculator amino acid class colour editor is described in Colourmap on page 606 and the sequence calculator panel colour editor is described in Using multiple viewing panels on page 617 Changing sequence calculator colours Chapter 25 Automatic graph labelling scaling and printing Chapter 25 Automatic graph labelling scaling and printing Chapter
112. in vehicles where the driver does not share the same air space as the equipment Procedure Use the following procedure 1 Contact your service centre and obtain a return authorisation number for your equipment 2 Complete the Equipment return repair declaration form and send it to your service centre The declaration must arrive at the service centre and permission to ship obtained before the equipment is despatched Sample records 20 Chapter 2 Safety warnings 3 Remove all traces of dangerous gases Pass an inert gas through the equipment and any accessories that will be returned to your service centre Drain all fluids and lubricants from the equipment and its accessories Disconnect accessories from the equipment Safely dispose of the filter elements from any oil mist filters Seal up all of the equipment s inlets and outlets including those where accessories were attached You may seal the inlets and outlets with blanking flanges or heavy gauge tape Seal contaminated equipment in a thick polythene bag If you do not have a polythene bag large enough to contain the equipment you can use thick polythene sheet If your equipment is a large pump or other large piece of equipment strap the equipment and its accessories to a wooden pallet Contact your service centre if you cannot meet this requirement If your equipment is too small to be strapped to a pallet pack it in a suitable strong box
113. informed of progress the file name of the data file being processed together with a measurement of completeness For example This is Run2xml Generating mzXML files 1 5 C data test0001 run Done 2 5 C data test0002 run 45 Warnings and non fatal errors If the application encounters an error which does not stop processing from happening processing you can pause processing while you answer any questions that the program requires to continue Alternatively the application may need to inform you of a problem If you used the y switch the software will always answer yes to any questions that the software might pose This will happen in the following circumstances Non Fatal Error Description The destination If you used the o switch but the directory does not specified output directory does not exist exist you must tell the software whether it should create the directory or whether it should exit The software will create all intermediate subdirectories if required The log file You have specified a og file which already already exists exists in the output directory the software will ask whether it should overwrite the file or just exit Table 34 5 Non fatal errors Using the command line editor 551 552 Chapter 34 Batch processor XML export Fatal Errors If any fatal problems occur during processing the utility will stop processing and report what has happened Where possible the program will a
114. ir is I p Insert Delete Mass Formula Calculate Monoisotopic Actual mass of the reference peak Calibrated mass range 1046 54 3657 93 1 missed references LS correlation 1 000000 Figure 27 11 Controls used for assigning observed peak masses 9 Press f4 to get the observed mass under the cursor from the display The Cursor mass will appear on the calibration window and be automatically inserted into the reference list 10 Repeat steps 8 to 10 for the remaining calibrant reference peaks A in the Cursor mass column of the list indicates that an observed mass has not been set In this case the observed mass is assumed to be the same as the reference mass 11 Press the Calibrate button If the calibration is successful the spectrum display will be updated to show the new mass assignments The calibrated mass range will be reported in the status bar of the base window A reminder message will be shown on the Calibration window about saving the calibration which is explained in the next section Should the calibration succeed but give an unexpected result press the Undo button this has the effect of returning the data to its previous state prior to calibration Calibration of the instrument Chapter 27 Instrument Calibration Note that is easy to toggle quickly between Average and Most abundant mass in the list box Hold down the right mouse MENU button over a selected item in the list an
115. is vented Isolates flight tube from analyser for door opening Shown as open when instrument is acquiring Instrument just switched on System is vented at atmosphere for maintenance Pumping down Flight tube Begin to pump down the source Pumping down SAC only Instrument is pumping ready for data collection Note The vacuum system shows System pumped when this pumping starts The system will not be ready to collect data until one minute after the turbo pump is up to speed and the vacuum gauge reads 2x10 Chapter 11 Checking the instrument status m Axima Confidence instrument status The instrument status diagram gives an overview of the instrument status in real time Instrument Status Tuning mode reflectron_ms Instrument mode Reflectron ee Door closed 45 33 82 88 xy Stage Pump Supplies Pressure Valves fow Po ee bew bes eoc Source f Lens Reflectro X motor At spee On OK Open pcradtion i Source Source Linear Accelerating Poor m SEN B oat natnchoi foe motor s off off Closed Reflectro Reflectroy Reflectron Inlet Fail Fail Fail X Defl Y Deti Detector me Figure 11 4 Axima Confidence status diagram The key at the bottom of the diagram indicates the possible states of the units Axima Confidence instrument status 97 Chapter 11 Checking the instrument status Axima Confidence status diagrams key The items on the diag
116. is being collected a number of profiles can be averaged together to produce one set of data This number is specified on the Data storage window see Storing collected data on page 146 The graphs can be automatically updated either after this average has been calculated or at the end of data collection for the current sample Setting Display to After average or End of sample provides flexible control over when the display s contents are updated For the Profile and Averaged graphs the data is displayed without any extensive processing taking place giving a rapidly updated display The Processed and Peaks graphs require the data to be processed during which smoothing of the data baseline subtraction centroiding apex peak detection peak labelling and other processes are carried out The labelling which appears ona graph can be set either to show as much information as possible or tailored to avoid clutter by choosing none some or all of Auto labels Peak Markers or Manual labels Automatic printing of results Automatic printing can be set so that the graphs are printed after calculation of the average or at the end of data collection for the current sample Set Print to After average or End of sample When the Print option is set to Off automatic printing is disabled Optional Features in Display Headings Most data displays begin with a standard heading which shows the following components see Figure 20 31 on page
117. is enabled and it is set using the gate masses field The field will accept either one or two mass values to be compatible with gating and blanking but in blanking mode only the first of these is displayed and used for the blanking function In the example in Figure 14 14 above blanking was enabled and the low mass value was set to 500Da The result is that peaks above 500Da are seen Blanking low mass ions Chapter 14 Collecting data from a sample Acquiring MS MS data on Resonance The basic mode of operation of the laser firing window on the Axima Resonance is the same as for the other instruments in the Axima series However there are some differences The window is shown below again for reference 4 Acquisition positive Firing Exp Tech Auto Quality Storage slide Raster Tuning QIT ToF ms T Auto quality a P Activate Accumulation Filename Profiles om per sample Shots fe xl accumulated per profile oe MSn Precursor Ions Mass Range PEHE 1005 3003 8503 2k Figure 14 15 Axima Resonance laser firing window MS Mode acquisition To acquire an MS mode spectrum on the Axima Resonance first select the mass range of interest using one of the 5 buttons in the centre of the window labelled Low 100 gt Low 500 gt etc These correspond to the 5 standard modes of operation that will have been set up in the factory Next ensure that the button labelled MS is in the off position Th
118. keyboard Ctrl key down while pressing the mouse SELECT button Release the mouse button and the graph will be redrawn expanding the range of profiles to that selected with the mouse Figure 17 13 Displaying Chromatograms 285 Chapter 17 Viewing the collected data lnt 100 2 0 mV mass 3715 to 14680 10000 Mass Charge ee Drag the mouse along the range of profiles to expand the range lnt 100 11 mV mass 1000 to 15000 Smoothed 1 7 z eS Gc x 100 50 a 7s 0 FSS 0 1600 180 2000 Profile Figure 17 13 Expanding the range of profiles on a 3D chromatogram Selecting a mass range on a 3D chromatogram Using the mouse move the mouse pointer to a position on the graph at the start of the required mass range Press and hold down the mouse SELECT button and drag the mouse diagonally along the mass range to the required end point of the range To force the mouse to move in one direction only hold the keyboard Ctrl key down while pressing the mouse SELECT button 286 Displaying Chromatograms Chapter 17 Viewing the collected data Release the mouse button and the graph will be redrawn expanding the mass range to that selected with the mouse Figure 17 14 lnt 100 2 0 mV mass 3715 to 14680 100 10000 Mass Charge Drag the mouse outside th
119. label maybe defined Opaque labels are useful to improve the legibility of the labels when they are put over a peak itself To set the colour of the label simply click on Set and choose the colour of the label using the standard Windows colour chooser A Font for the label text may be selected from the drop down list A Size can be specified in units of percentage x 10 of the window height The label may be rotated by setting the Angle in degrees Bold Italics and Underline properties may also be set for the label by checking the relevant boxes All changes are applied when the OK button is selected Note that for user defined labels i e manually entered rather than software generated multiple selection of labels on a graph is possible In this case the Annotation Properties option on the graph MENU is enabled If this is selected then the Annotation Properties window again appears but in this case the properties such as font angle etc are applied to all of the selected labels Chapter 20 Managing Data Displays Panning displays Panning may be used to locate data quickly and to make fine adjustments to the mass profile ranges displayed Panning is the ability to obtain a panoramic view of the whole data by moving from one end to the other easily There are different modes of panning spectral displays which are outlined in the following sections Repositioning a peak in the selected display This is achieved by h
120. load data toolbar button Exit Figure 8 1 Load data toolbar button In both cases the Load Data window is displayed this window is used to select and load previously collected data Figure 8 2 Load Data Oix o Data set No rpn O J pois Im I le l In OK Browse Unload all Figure 8 2 Load data window Loading data ecocoooo N uo 76 Chapter 8 Loading and unloading data To list all available datasets which have been stored press the Browse button This will display the Data Browser window Figure 8 3 e Data Browser BEI Folder c program files shimadzu biotech launchpad data bill p Shimadzu Biotech Launchpad Fiter Options Find Dataset E Calibration Mode Any x P Ext Any hd praia Polarity Any 7 Name f ny x Fin 5 ata 04 March 07 Mass Any 7 Title Any gt E 8 4 02 lon Selection Confirmat E Andy Gate Any Comments Any zi Look in Sub Folders I C Beam blanking 7 Display Options ba ii 1 Bill of Sont by name 7 Display Title line ba List Dataset ate Mode Gate PEt Title dh ang11__0001 2007 02 03 Blanked ah angio 2 with theoretical0001 2008 01722 Blanked Jll angio 2 with theoretical0002 2008 01 22 Blanked dh bill_0001 2008 01 22 Blanked dl cal_ooo1 Blanked ME psd_angi1_0001 200 Blanked Ll psdcal__0001 Blanked Figure 8 3 Data Browser window The Folder list shows all folders in the user
121. m Headings X Axis M Y Axis V Display Heading IV X Aris Title V Y Axis Title MW Trace Headings IV Separate X Axes I Markers Baseline V Peak Limits Threshold V r Sealing Profile Scale Automatic Isometric plats Angle fas a Base height 50 m Colour editors Spectrum Chromatogtam Distribution Comman I All displays Figure 25 2 Display Options window Graphs page Marking the subtracted baseline If Subtract Baseline from signal has been selected on the Processing window the level of the signal baseline which has been subtracted from the total signal can be displayed on the averaged graph by ticking the Baseline marker option on the Graphs property page Markers displayed on a spectrum 445 Chapter 25 Automatic graph labelling scaling and printing This gives a clear indication of the extent of the signal being removed from the data Figure 25 3 Baseline being subtracted is shown on the average trace lnt 100 8 mV sum 813 mV Profiles 0 100 Averaged 100 504 0 lnt 100 6 mV sum 637 mV Profiles 0 100 Smooth Av 10 Baseline 40 5686 0 100 i 50 5650 5700 Mass Charge Data after baseline subtraction is shown on the processed trace Figure 25 3 Baseline subtraction Marking the limits of peaks When peaks are detected on spectra theoretical distribution and reference profile graph
122. mzXML MZXML File E 3 My Documents 75 00 07 29 1 109 Im20001 mzXML MZXML File O Batch processor results i E 76 00 07 35 1245 0001 mzXML MZXML File imaging output 76 00 07 35 1245 Imz0001 mzxML MZXML File E O iraq 3 79 00 07 57 1636 0001 mz XML MZXML File LC MALDI E 79 00 07 57 1636 Imz0001 mzxML MZXML File My eBooks E 80 00 08 03 1047 0001 mzxML MZXML File My Music E 80 00 08 03 1047 Imz0001 mzxXML MZXML File 3 83 00 08 21 1339 0001 mzxML MZXML File gt LIA E 2 my Pictures ELI Mw Proiects O File conflicts You will get a file conflict message if There are two files with the same name but in different directories and e You attempt to convert the files to the same destination folder Otherwise you could overwrite a file from another experiment Processing settings Data files include the peak processing parameters 2 7 and later which are required when exporting to mzXML and mzData If required you can apply a different set of peak processing parameters when exporting the data If you have old data files generated using previous versions of MALDI MS software prior to 2 7 they will not contain peak processing parameters You can add these parameters to the legacy file Using the Batch processor Chapter 34 Batch processor XML export 1 Select the Processing Settings button You can apply a different set of parameters to the exported data The selected setting
123. navigate to the directory where the run file exists 2 Type the command run2xml 54 00 05 28 1167 0001 run mzxml Multi file conversion example To convert all the run files in a directory to mzdata format type in the command 1 In the command line editor navigate to the directory where the run files exist 2 Type the command run2xml run mzdata List of Command line switches The following tables in this section are a list of all the command line options that the program will respond to Examples of how these switches fit together are given later Input files are specified by entering the name or names of files in a list The and wildcard characters are accepted as part of any filename and will be interpreted in the recognised manner Any command line arguments that does not begin with a forward Slash will be regarded as being a specification of a filename There is no required order of arguments Using the command line editor Data format mzxml mzxml raw mzxml process mzxml peaks Chapter 34 Batch processor XML export Export mzXML default type is raw data Export raw data in mzXML Export processed data in mzXML Export peaks in mzXML mzdata Export mzData Table 34 1 Data format Input Output folders i dir Set input directory If left blank input files are taken from the current directory If the directory includes spaces place quotes around it o dir Set outp
124. number of lines in the display increases the font size and increasing the number of lines will conversely decrease the font size The Lines to print option allows the number of lines printed in a text report to be controlled This can be adjusted to suite individual requirements in either portrait or landscape printing mode Customising Text Reports 381 Chapter 20 Managing Data Displays Annotation Graphical displays can be annotated using a selection of drawing tools Facilities are provided for placing text anywhere on a display and drawing lines and boxes over data Select the dataset to annotate by clicking on the Process button for the dataset to be processed on the Spectrum Contents window To add annotation select the Annotate option from the Display menu as shown in Figure 20 36 Spectrum Contents Se x Dataset Trace Sample Process 1 angl1__0001 Scroll dataset Process Peaks e Set 15 610 Display multiple samples Select dataset for processing New Annotation ME E Annotate Tat EES m ksd Annotation Properes Tag Decimals 4E nm angio2_msmst alli Peak labelling Figure 20 36 New Annotation window 382 Annotation Chapter 20 Managing Data Displays The coloured square adjacent to the dataset name indicates the colour of the dataset trace to which annotation will be applied To add anything other than text both graph cursors must be shown
125. of camera fitted By default this will be BW CCIR which is the type for the black and white camera normally shipped with the Axima When the mouse pointer is moved over the camera image it changes to a special cross hair pointer jue PrE ll A This pointer can now be used as alternative means of moving the stage Simply click the mouse SELECT button over the desired point of laser impact and the stage will automatically move to position the normal red cross hairs which indicate the focus of the laser over the point of selection Using the camera Chapter 14 Collecting data from a sample Blanking low mass ions The Aximas are equipped with low mass blanking hardware in order to improve the sensitivity of the instruments This is achieved by suppressing the Matrix ion current before it reaches the detector T2_mix_blanked_500Da0001 T2_mix_unblanked0001 MALDI MS File Edit view Instrument Automation Processing Help T2_mix_blanked_500Da0001 T2_mix_unblanked0001 Shimadzu Biotech Kompact MALDI 6 V2 7 0 Beta 1 alnt 493 mV 1416 mV 1046 54 Spectrum not blanked 1046 50 379 09 600 Mass Charge For Help press F1 Figure 14 14 Blanking example Blanking low mass ions ecccce a 160 Chapter 14 Collecting data from a sample If this hardware is available it will appear as an extra button on the laser firing window next to the ion gate and labelled Blanking When selected the hardware
126. of 1 or 2 hydrogen atoms Loss of 17 Da Probably due to loss of NH3 lons produced by side chain fragmentation lons produced by side chain fragmentation 625 626 Chapter 42 Sequence Calculator Introduction Johnson R S Martin S A and Biemann K Collision Induced Fragmentation of M H lons of Peptides Side Chain Specific Sequence lons Int J Mass Spectrom lon Processes Vol 86 Pp 137 154 1988 e Stults J T and Watson J T Identification of a New Type of Fragment Ion in the Collisional Activation Spectra of Peptides Allows Leucine soleucine Differentiation Biomed Environ Mass Spectrom Vol 14 Pp 583 586 1987 The diagram below defines the ionisation fragments using the Biemann notation It is also common in MALDI techniques to observe A 17 and B 17 ions which may be due to the loss of NH3 but at present there is no conclusive evidence as to the specific nature of the group causing this mass loss lons in the m series are formed by the loss of one of the side chains from the complete peptide fragment lons in the i series are internal immonium ions Reference 5 The d and d ion series are equivalent to the d ion series reported by Johnson Martin and Biemann Reference 6 Two columns have been included to allow for the fact that certain amino acids such as Isoleucine give two d ions Other amino acids such as glycine do not give d i
127. of the laser This wheel varies in its opacity in 180 steps where 0 is minimum transmission and 180 is maximum transmission You may choose a fixed power level by entering a value after Power or by adjusting the slider or choose to step through a range of power settings by entering a range of values after Power e g 30 80 The old button chooses the mid power level and the j j button sets the power to ramp automatically from lowest power to highest The laser power can be adjusted while data is being collected The optimum laser power can be estimated automatically by ticking the Auto quality option See Automated data quality filtering on page 153 which describes the auto data quality feature however it is suggested that basic instrument operation is studied before using this feature Setting the laser power 45 146 Chapter 14 Collecting data from a sample Storing collected data Very often a large amount of data can be produced whilst simply finding the sweet spot on the sample slide If data were stored for every profile collected from every sample the computer s hard disc would rapidly run out of free space To this effect some consideration should be given to whether data storage is really necessary Many profiles may contain little or no valuable data To store these profiles would be extremely wasteful of space on the computer s hard disc For this reason a number of features have been provided to reduc
128. of the parent mass The following procedures assume that you are familiar with MALDI MS software and ion fragmentation You will need a sample of P14R Chapter 28 Fragment ion calibration m Axima Performance The aim of the fragment calibration procedure is to define as accurately as possible the relationship between the apparent mass and the actual fragment mass Moreover this must be done for the mono isotopic masses peaks only the isotope peaks corresponding to no C carbon or only C isotopes in the ion the lowest mass isotope in the distribution Apparent mass Actual mass Fragment varies between mono isotopic peak instruments only y14 1469 1436 81 y13 1405 1339 75 w12 1309 1199 66 wll 1242 1102 60 w10 1174 1005 55 w9 1104 908 50 w8 1034 811 45 w7 961 714 39 w6 836 617 34 w5 808 520 29 w4 728 423 24 w3 644 326 18 b2 521 195 11 b1 422 98 06 Table 28 1 Fragment calibration peaks for P14R Axima Performance The table above contains a list of the fragment peaks for P14R The name of the fragment is in the first column and its correct mono isotopic mass Actual mass is in the third The middle Axima Performance 483 484 Chapter 28 Fragment ion calibration column Apparent mass contains the approximate mass at which the fragment peak appears when the fragment calibration is not applied i e when the normal calibration is active Data p14r_msms0004 13 c 28 Sep 2005 13 35 Cal fe_test
129. on the Note Contents window The modified note file will be shown in the selected display Displaying previously created notes Use the Browse button to display a list of notes which have been written for the current data This window Figure 17 18 shows the first line of each note which has been written ba Browse Notes Be x Note Contents Calibration information and list of suitable references Figure 17 18 Browse Notes window Select the note which you wish to display from this list using the mouse SELECT button This will set the note number accordingly on the Display contents window Finally press the Apply button to see the note Other notes Any ASCII text file can be imported into a display and this is done by using the Other notes file option These files are not note files and as such are not stored with the data This feature may be used to display standard laboratory reference information such as the name address fax and email address of your lab alongside data without the need to duplicate this information with each piece of data collected Displaying laboratory notes 291 Chapter 17 Viewing the collected data To display any text file as a note set the note type to Other and press the List button a file selector window will appear allowing the text file to be selected for display Select the file from the list and press Open Figure 17 19 Select Notes File 21 x Look in e Shimadzu Bio
130. on the toolbar Display Contents button to show the Distribution Contents window Figure 23 1 Distribution Contents iOi x Formula Scale Adduct1 M jo a Gain Loss Adduct 2 M jo a Gain Loss Adduct 3 M jo a Gain Loss Adduct 4 M jo a Gain Loss Simulated resolution 2000 a Auto mass range V Clear Figure 23 1 Distribution Contents window Distributions can be calculated for a molecule with up to four adducts By adjusting the scale factors different ratios of the various adducts can be simulated The simplest example of a distribution is to show a molecule without adducts The example which follows shows the distribution for 5 tin Sn atoms simulated at 2000 resolution measured at 50 peak height To create the example 3 Set the parameters as shown in Figure 23 1 Displaying isotopic distributions 2417 418 Chapter 23 Displaying simulated data 4 Press Apply on the Distribution Contents window Molecular formula Sn5 Resolution 2000 at 50 olnt 100 672 52 577 52 0 595 Mass Charge Figure 23 2 Distribution for Sn at 2000 resolution The program automatically calculates which isotopes of the molecule will occur and in what ratios Each isotope is simulated by a Gaussian shaped peak having the required resolution For more complex formulae the number of possible isotopes can run into millions The program automatically selec
131. perform a cyclic calculation the peptide sequence is entered in the usual linear manner but with no c or n terminal groups specified on the main Sequence calculator window As no terminal groups are specified bonding between the terminal amino acids lysine K and glutamic acid E is assumed Cleavage 4 K R E t A L T Cg O 4 tR S I C as can be seen results in only two fragments ATOSCIR and CLEKR For the purposes of illustration we assume here that the normal enzyme cleavage rules for linear sequences still apply and that the n to c terminal direction is clockwise even though neither n or c terminii exist in a cyclic peptide As can be seen Chapter 42 Sequence Calculator the order in which the amino acids are considered for a cyclic peptide represented linearly is very important Here if we represent the sequence as n CLEKRIATOSCIR C with the cleavage now as shown the correct fragments are found The sequence calculator automatically rotates the sequence order for cyclic peptides to ensure that correct fragments are encountered Clearly however the truly linear sequences KRATOSCIRCLE and CLEKRATOSCIR are different so caution should be exercised if sequences are saved to a database after cyclic calculations have been performed Contents of the peptide reports The content of the reports reflect the type of fragmentation selected An example of an enzyme digest report is shown in Figure 42 25 terminus
132. place if one of a specific set of amino acids is adjacent to the cleavage site The adjacent amino acids in both cases are specified following the Adjacent to option Up to nine amino acids can be entered As an example defining an enzyme to cleave on the C Side side of K with Not Adjacent to AL would cleave as shown in Figure 41 3 N terminus I A K A P K L A K P A Q C terminus No cleavage ae cleavage site Figure 41 3 Example of an enzyme cleavage definition After defining a cleavage rule it can be added to the list by pressing Insert To remove a cleavage rule from the list select the cleavage with the mouse SELECT button and press Delete Chapter 42 Sequence Calculator Chapter 42 Sequence Calculator 602 Chapter 42 Sequence Calculator Introduction The Sequence calculator supports a comprehensive range of calculations required for structure validation In addition it enables users to create and maintain a database of peptide sequences for direct comparison of peptides and enzyme digest products with collected data It can read and display peptide fragments from commercially available databases in fasta e g NIH database and seq format e g EMBL database The Sequence Calculator is started from the Processing menu as shown in Figure 42 1 X pi4r_msms0004 MALDI MS Pile Ea File Edit View Instrument Automation Processing Help ielai ol Calibration Slane Te leo
133. s ssssssssnsunnnnnnnnnnnnnnnnnnnnnnnnnn 559 Element database cccccceeeeceeeeeeeeeeeeeeeeeeeaeeeeeeeeuaeeeeeeeeeeaneeeas 563 Accessing the database cc cc cceeee cece ee eee eee tenet nrerin rnern 564 Creating a compound database scceeeeeeeeeeeeeeeeeeeeeeeeueneaeanes 569 MEMOGDUCTION shied fe escdansd Sains ew ob ia heap TE E weedeat anne 570 Searching for molecular weight Matches cceeseeseeeeeeeeeeeeee 583 Polymer simulation ccccccceceeeeeeeeeeeueueeeeeeeeueueueuaeeeseuereeeeeeeenens 589 INErOdUCION eeccs ccd ce eee cee sees ee oeee ceed e nde eeeue ea oedau ied ieeey eemdewceten 590 Defining ENZYMES sesiisccciiececsciwicet cescad esenencsceened casein seedemeeew snes 597 SEQUENCE Calculator sivscssiicccccsseccccsccssccssssseidisasscedtesnsededdennaeeds 601 PMUPOGUCHION 6 ai stavidded cds e dec nie sataw le hile ETE a e 602 Listing of the template itn file sssssssssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 643 Summary Of error MESSAGES cseseeeeeeeeeeeeeeeeeeeeeeeeeeeeeeoeeeeonenens 647 Introducti OR ixises oc tas cet idteined caeeeiaied dant grees ene ones Leaded 648 Etror messages ssrin esse venan dings peed aming sh indea dana release aaia 649 Warning messages cceee cece eee eee eee teat e etna teeta tate 660 BibliOQGraphly ccccceeceeeeceeeeeeeeeeeeeeeeeeeaeeeeaeeeeeeeeeaeeeuaeeeeeeeeueeeanans 663 Acknowledgement ccccsceeeeceeeeeeeeeeeeeeeeeeueeeeeaeeeuaceaeeauageauaneas 665 Chap
134. selected in which case a new file will be created When suitable parameters have been entered in the window they should be saved to the method file The Print Spectra check box if selected causes the spectrum to be printed with the mass range rescaled based on the primer and loss mass peaks encountered for the sample during the oligo analysis Note that this is in addition to any spectral printing which might be specified in the method s Output Print options see Print on page 194 The experiment is run in the normal manner from the Auto Experiment window The output file produced might be as shown in Figure 15 23 below Al ZUS24a PASS 10521 4 286 7 4090 7 20 8 3091 4 12 6 B1 ZUS24b FAIL 12345 6 no peak 5915 2 19 5 4915 3 10 6 C2 ZUS25c UNCERTAIN 15431 9 300 1 9001 4 21 1 8000 5 11 9 sample primer peak i source ID mass and area loss peaks mass and area sample test result well ID Figure 15 23 Oligo Analysis ASCII output In this example sample A1 has passed because the primer and both loss peaks were detected within the tolerance the loss peak areas were both within their respective percentage of the primer peak area the sum of the loss peaks area was less than the lower percentage limit of the primer peak area and lastly because the signal to noise ratio was acceptable Sample B1 has failed because no primer mass peak was detected Sample C2 was classed as uncertain this was likely du
135. source and final destination folder are transparent to the Introduction 521 522 Chapter 32 Archiving data Archiver For example reference files will be copied from and written to the reference file folder regardless of its location on either computer system After restoring the selected files click on the End restore button this will put the window back into Archive mode and display the contents of the Shimadzu Biotech Launchpad folders To restore archived data from a file use the same procedure as outlined above simply select the file on the fixed disk or network drive when the window shown in Figure 32 7 appears Using the Archiver to compress files Introduction The Archiver window can be used to compress files so that they take up less disk space This is performed using gzip compression which gives a consistently high compression performance ratio over other compression algorithms To compress files select the files as if they were to be archived in the same manner as described in Archiving to removable disk media on page 519 and then press the Compress button All of the selected files will be compressed This is useful for files which are not used often or for data files which are uncompressed when loaded but reference label calibrant reference and parameter files cannot be used by MALDI MS software if they are compressed Compressed files appear with a compressed icon i e a G clamp surrounding the normal
136. started The Launchpad Each of the programs on the Launchpad menu can be started by selecting the respective menu item However there is another way using the dedicated Launchpad program This is started by selecting Launchpad from the Shimadzu Biotech Launchpad menu see Figure 3 6 The Launchpad shown in Figure 3 7 will be displayed This window has all of the commonly used program icons on it and provides a quick means of starting the programs Simply click on the icon of the program that you wish to start Clicking on the I nfo button will display the release notes issued with each release of software These notes will point out any updates or fixes from the previous release of software Programs ci oon 4 Compounds Database se Favorites gt g Configure Environment L Im Accessories gt 3 Element EG Boomers f Administrative Tools Element Database Editor ee Settings Nai Launchpad 2 7 Enzymes g IT Quadralay gt Kompact Search RoboHelp Office aa Launchpad Help and Support SmartDraw 4 ee konno IT Soundmax gt SS Polymers Run f Startup gt FRE Sample Plate Editor i Symantec Client Security gt Search Log Off bill IT Volo View Express y Turn Off Computer Figure 3 6 Starting the Launchpad from the programs menu The Launchpad Chapter 3 Getting started Launchpad Of x Show on startup V _Heb APE MALDI M
137. started guide Chapter 2 Using the Axima Launchpad amp MALDI MS section Multiple tiles The following buttons usually on the right hand side of the display allow you to manage the display Button Function Zoom width with two or more columns expands tile to full width Zoom height with two or more columns expands tile to full height Full window expands tile to full window size Using Auto Experiment Results viewer 231 232 Chapter 15 Automated operation Button Function g Full window expands the next tile to full window size lt Refresh will re read the currently loaded results 5 file if it has changed ET Output results use this button if you wish to EE save the experiment results to a text file The file is a tab delimited text file which is compatible with spreadsheets Using Auto Experiment Results viewer Chapter 15 Automated operation References 1 Lechner D Lathrop G M and Gut G Large scale genotyping by mass spectrometry experience advances and obstacles Current Opinion in Chemical Biology 6 31 38 2001 2 Sauer S Lechner D Berlin K Lehrach H Escary J L Fox N and Gut I G A novel procedure for efficient genotyping of single nucleotide polymorphisms Nucleic Acids Research Vol 28 No 5 e13 2000 References 233 Chapter 15 Automated operation 234 References Chapter 16 Cleaning up data Chapter 16 Cle
138. the data storage window Compressing data that is active can significantly slow down the process of loading and unloading datasets The Windows operating system has its own file compression facility which can be enabled for a folder as shown in Figure 14 9 below The old data folder to be compressed is highlighted with a right mouse click and the Properties option selected Now select the Advanced button from the Properties page and this allows the compression option ticked in Figure 14 9 to be selected in the Advanced Attributes page Additionally changes in the cost and capacity of computer data storage over recent years means that file compression has become of less importance for most users In the interests of good housekeeping however it is still important to archive older datasets which are no longer active and the Archiver program still has facilities to compress and decompress datasets Any compressed files will still be automatically de compressed if they are loaded for viewing or re processing Averaging profiles Chapter 14 Collecting data from a sample Calibration Comments a G ex int E Shimadzu Biotech Launchpad Data Properties Advanced Attributes Figure 14 9 Compressing a folder in Windows Averaging profiles 149 150 Chapter 14 Collecting data from a sample Firing the laser Having made the necessary selections on the Laser firing windo
139. the data set selected on the Calibration window and wish to load an existing calibration and apply it to this data select List for Selected data set and choose a calibration On the Calibration window set the Load option to Named calibration and press the Load button to apply the chosen calibration to the selected data set The procedure for calibrating the instrument under normal conditions has been described However there are optional calibration features which may be important in particular circumstances The first of these is the ability to create a calibration from calibrant samples on separate sample spots or even separate sample slides Calibration of the instrument 475 476 Chapter 27 Instrument Calibration Combining calibrations from different sources The situation may well arise where two or more calibrant compounds may be useful to span the required mass range but these cannot be prepared on the same sample slide In this instance a mechanism has been provided to allow the user to combine calibrant peaks from different sample slides The method for combining these peaks is described below 1 First acquire all of the data sets which are to be combined using the same instrument calibration for each sample acquired 2 In the Load window load the data which is to be calibrated and all of the data sets which are to be combined to create the new calibration 3 Ensure that the data to be calibrated is the current
140. the element first followed by the number of that element For example CgHs would be entered as C6H5 Any formulae or compounds entered in the compounds database can also be used in creating reference files see Creating a compound database on page 569 Select either the Calculate Average or Most Abundant or Monoisotopic masses button to calculate the elemental mass and display it in the Mass entry A letter M will appear in the Abundance column of the table for masses which are entered as Most Abundant masses and a letter A for masses which are entered as Average I is used to indicate Monoisotopic masses Selecting Most abundant calculates the most abundant isotope combination e g Cgo is calculated as 1 Cgg13C Likewise Average calculates the average of the isotope masses weighted by natural abundance Average isotopic mass will give a closer representation of the centroid mass of a cluster of completely unresolved peaks Monoisotopic masses use only the mass of the most abundant elements in calculating the molecular or formulae Creating a new reference file 463 464 Chapter 27 Instrument Calibration masses For smaller molecules the monoisotopic and most abundant masses are likely to be the same i e the lowest mass peak in a distribution but for large molecules the approximately 1 of 13C become more significant and the monoisotopic and most abundant masses will differ Compounds created using the Comp
141. the whole mass range for the selected sample as extrapolated from the calibration The mass range can be rapidly scrolled by ten percent in each direction by clicking the mouse SELECT button on the display toolbar right and left arrows A horizontal scroll bar at the bottom of graphical displays is also a useful means of rapidly moving backwards and forwards across the X axis of the currently selected graph An even faster method of selecting a portion of the displayed mass range is to use the mouse Move the mouse pointer to a position on the graph at the start of the required mass range Figure 17 5 Press and hold down the mouse SELECT button and drag the mouse horizontally along the graph to the required end point of the mass range Release the mouse button and the graphs will be redrawn expanding the mass scale to that selected with the mouse Figure 17 6 Displaying Spectra 273 Chapter 17 Viewing the collected data File Edit View Instrument Automation Processing Help w ha Se 2 al x lt Display Spectrum Profiles 1 Masses 1272 1323 Kiil Data pep_mix_2466d0005 110 c 10 Feb 2005 10 06 Cal tof 10 Feb 2005 9 02 Shimadzu Biotech Kompact MALDI 6 V2 7 0 Beta 1 Mode reflectron_ms Power 45 Blanked P Ext 2460 bin 4 Yalnt 1 6 m sum 220 mv Profiles 1 142 Smooth Av 20 Baseline 80 1297 18 100 Glick and hold the Sweep over the area of left mouse button interest and release 1300
142. they are discarded See reference 1 below for a more detailed description of the Poisson modelling algorithm used Chapter 16 Cleaning up data Filtering specified peaks In addition to the functionality of the Peak clean up window just described the user may wish to define a range of peaks that are to be filtered from the reported peak list For example it may be useful to ignore peaks that are due to the matrix or to ignore all peaks below a specified mass prior to peptide mass fingerprinting Filtering specified peaks 259 260 Chapter 16 Cleaning up data The parameters needed to perform peak filtering are found on the Filtering tab of the Peak clean up window see Figure 16 12 on page 260 Note that the filters are only applied if the Peak filtering check box at the top of the main dialogue is ticked MpPeak Processing BEI Dataset fil 1 angio2_msms0006 Ne Trace i Sample G7 Peak Cleanup Monoisotopic Peak Picking Peak Filtering Peak Filter Editor Cal Flag lt 5670 gt 5810 v 5720 6 74 Selected Filter Append Delete Clear Tolerance 0 5 Da ka Convert formulae as Average masses ii Load Save Filters from Peaks Apply to Tiles l Hal Apply to Samples E yW Iso Mono Figure 16 12 Filtering tab window The window serves two main purposes the main portion is used to define the filters and filter tolerance to be applied the lower smaller portio
143. to zero Labels may be rotated by setting the Angle in degrees The Size of the label may also be specified in units of percentage x 10 of the window height Bold I talics and Underline properties may also be set for the label by defining the relevant check boxes lnt 100 6 mV sum 643 mV Profiles 0 100 Smooth Av 10 Baseline 40 100 80 60 40 20 5685 96 5816 84 5599 93 5773 34 5700 5750 5800 Mass Charge 5600 5650 Figure 25 7 Mass Peak labels 2 decimal places Difference and Relative labels can also be applied to collected data The Difference labels show the mass difference between each labelled peak The lowest mass peak is labelled with the actual peak mass All other peaks are labelled with difference from the last labelled peak Figure 25 8 Labelling peaks 452 Chapter 25 Automatic graph labelling scaling and printing lnt Labelling peaks 44 25 5512 85 5550 42 83 43 50 5600 5650 Mass Charge 100 6 m sum 637 mV Profiles 0 100 Smooth Av 10 Base 4 ine 40 2 53 44 43 5700 Figure 25 8 Polymer spectrum with Difference labelling Using Relative labels allows the differences to be shown relative to a selected peak To use relative labelling set Label type to Relative On the display press the mouse ADJ UST button and move the mass cursor over the peak to be used as a reference point Release the mouse button and c
144. two columns one for masses one for the corresponding tolerances The fields must be separated by either a tab or a comma and the rows separated by a carriage return Mass and tolerance values are in Daltons An example text file follows Column 2 Column 4 mass data tolerance 20 1600 xyz 2 30 2500 abc 1 40 5750 def 2 50 8030 ghi 2 In the above example you set lon Finder to separate fields using commas e define mass data in column 2 e define tolerance data in column 4 After importing the text file you can subsequently edit the masses and tolerances Clipboard data The data pasted from the Clipboard contains the target ion mass and target ion tolerance in the same format as described above The rules for importing data i e field separator and input field numbers are the same as described above Importing a list of masses Chapter 19 lon finder Importing data 1 5 6 From the lon Finder window select the Settings button the lon Finder settings window is displayed Ion Finder settings x p Import columns C Mass column Cancel Mass tolerance column 2 Import field separator Tab Comma Reported peaks All peaks C Largest peak in range Nearest peak in range Required decimal places 0 Figure 19 3 lon Finder settings window In the Import columns fields enter the column numbers corresponding to the mass and tolerance data within the text
145. will apply to all data which is exported These options can also be used to export data which was created in a version of Launchpad prior to 2 7 Use the set of parameters contained in the dataset Use the current parameter set from MALDI MS Use a specific parameter file Figure 34 6 Processing Settings window 2 Select the required radio button Use the set of parameters contained in the dataset Peak processing parameters are generated using the original instruments default values Use the current parameter set from MALDI MS Peak processing parameters are generated using the tof parameters file see page 80 for details about this file e Use a specific parameter file Peak processing generated are provided using a saved parameter file see page 81 for details about these files 3 If you selected the Use a specific parameter file option a Select the Browse button Open BEI Look in jo Parameters a fa My Recent Documents Desktop a My Documents a My Computer w My pew File name l z Places Files of type Parameter Files tofparams z Cancel A linear peptide tofparams E reflectron peptide tofparams Using the Batch processor 545 546 Chapter 34 Batch processor XML export Figure 34 7 Open window showing tofparams files b Navigate to the required tofparams file usually within the Parameters folder c Program Files Shimadzu Bio
146. will be written to the named file All comments files have the file extension comm Introduction Chapter 10 Putting comments with collected data Loading files To reload the comments at a later date Click on the Load file button select the folder and filename in which the comments were stored using the Select Comments Files window and press the Load button The contents of the stored comments file will be copied into the Comments window ASCII comment files You can create a Comments file using a text editor for example Notepad and load the file using the Load button select txt files in the Files of type field The ASCII comments file must be comprised of one line per comment where a line should consist of the sample well ID and the comment separated by white space Two special identifiers are also recognized in place of the well ID either PREFIX or TITLE can be entered The lines of text do not need to be entered in any particular order A fragment from such a file might be as shown in below TITLE Samples from batches 2 amp 3 using method 44 AlGome comment text associated with AT gt 7 Oiext belonging to well C2 sample comment sample comment to go with well B1 text well ID PREFIX Plate ID 18 Figure 10 3 A Fragment oe possible ASCII comments File Introduction 89 90 Chapter 10 Putting comments with collected data Introduction Chapter 11 Checking the instrument status Chapter
147. with Shimadzu or Kratos otherwise the Axima PC may fail Backup data Regularly backup data off the Axima PC to secure media We do not accept any liability for loss of data Enter one byte code alphanumeric characters For Far Eastern countries The Axima PC uses an English language operating system When entering characters and numerals use one byte code alphanumeric characters only Physical shock or vibration Avoid physical shock or vibration as the laser ion and or camera optics may become mis aligned Also the turbo pumps may be damaged Tilting the Axima Do not tilt the Axima as the turbo pumps may be damaged The Axima will fall over if tilted beyond 10 to the vertical Sample door Only use Shimadzu approved Target plates in the Axima sample door otherwise you may damage the Axima Target plates Always ensure that your samples are dry before putting the Target plate in the sample door as a good vacuum may not be maintained resulting in damage to the Axima Sample records Always keep a record of all samples used in the Axima Use the forms provided in the Customer Support Guide Health and safety precautions Chapter 2 Safety warnings Safety statements and warnings Please read this section of the manual carefully as it sets out all of the safety issues relating to the operation of the Axima range of instruments Ensure that it has been read before operating or carrying out any maintenance on the instr
148. x ak Display Reference List Profiles 1 54 X Masses 1044 3678 l gt I Reference mixed_ref r Mass Formula 147 1309 C5H9NO4 377 5259 C22H35N13011 956 1157 C44H69N13011 1253 4300 APGDRIYVHPF For Help press F1 dM 0 99 M dM 1061 26 CAP NUM Figure 23 11 Example of a Reference list report The reference data displayed is chosen in the same way as explained above for the reference graph using the Display contents window The Display contents window for a reference list is shown in Figure 23 12 3 Reference List Contents Bm es Data from Eae a Figure 23 12 Display contents window for a reference list 428 Displaying reference files Chapter 24 Choosing user defined colour schemes Chapter 24 Choosing user defined colour schemes 2429 Chapter 24 Choosing user defined colour schemes Introduction The MALDI MS software suite allows the user to change the colours of a large number of items in the displays including spectrum trace colours cursors and chromatogram trace colours These are all found on the Display Options window Figure 24 1 J default MALDI MS olx File Edit view Instrument Automation Processing Help lil v Toolbar h2 a lt Display Spectrum X Profiles Masses 12 1040 i gt i Ee xie Kij v Display Toolbar v Basic Parameters Display Options x A e me General Graphs Graph Text Text Report Cursors Peak Labels Options
149. y Copy comments jern gt M nao hean Clear comments Lood He Sve tie O C ox__ r _corcet_ Figure 5 2 File menu File menu Chapter 5 Window and menu guides File loading sub windows Accessed by File gt Open this is described in Loading and unloading data on page 73 Load Data SBE Data set X OE 0001 w Data Browser Folder c program files shimadzu biotech launchpad data bill cy Shimadzu Biotech Launchpad prior Options Find Dataset E Calibration Mode Any ad P Ext Any 7 ota Bolaty ary z Name ary z Data M l i 7 C 04 March 07 Mass Any fi Title Any 8402 lon Selection Confirmat fjw Day Gate Any T Comments JAn Look in Sub Folders a Beam blanking T Display Options Ca Bil w Sottfby name m Display Title ine F Jl ang11_0001 2007 02703 Ref Blanked 2300 Jl angio 2 with theoretical0001 2008 01 22 Ref 1050 lL angio 2 with theoretical0002 2008 01 22 Ref 1050 2008 01 22 Ref 2300 2008 01 22 lll psdeal_0001 2008 01 22 Figure 5 3 Load data window File menu Chapter 5 Window and menu guides Comments sub menus Accessed by File gt Comments this is described in Adding comments on page 86 for details Comments Be x Current comments E astaset 1 bitangio 2 with theoretioai0002 Title Prefix Select well List All entries Samples Comment o ooo o
150. zero will prevent the respective MS MS and Mascot searches being carried out If a Mascot search is not carried out then the number of MS MS is determined by Max Confirmation Peaks The Good PMF Score field is used to decide if confirmation MS MS are carried out If the first best PMF hits score is greater than Good PMF Score the confirmation MS MS are not carried out Chapter 15 Automated operation Auto Experiment Auto Experiment provides the facility to automatically analyse and process samples Processing involves acquiring the mass spectral data and processing that data by methods developed using the Method Editor Figure 15 32 shows the Auto Experiment window Facilities to edit save and load experiments are provided however only one experiment can be active at any one time It is started by selecting Auto Experiment from the MALDI MS Acquisition menu Auto Experiment Ele x Plate Experiment sei 7 QQ 4 Osi Bal 700 n wells Experiment file name not set Experiment Selection Selection Detail Comments and Results j ee Tl Results File E mail Notification I Upon completion of this experiment send an e mail to the address specified below b amples Type Stat End_ Samples Method DaaFie Comments Balx Figure 15 32 Auto Experiment window An experiment consists of a sample plate created using the Sample Plate Editor and groups of sam
151. 0 Overview E VLE Calibration pe Peak Cleanup Taxonomy Aeris 0 A Monoisotopic Peak Picker Enzyme fmn z Missed Cleavages T VLE Peak Filtering ypsin Z Ea Mascot Fixed Acetyl K L E Applications Modifications Acetyl N term Acetyl Protein N term gt Compare Sequence Amidated C term Oligo Analysis AE Output Variable Acetyl K AQ Print Modifications Acetyl N term 3 Expott Acetyl Protein N term I Report Amidated C term i lon Finder Max Peaks 20 Protein Mass kDa 0 ME MSMS Mass Types Monoisatopic Mass Values MH C Mr C MH VY Acquisition c amp Peak Selection Average A Raster Peptide Tol 1 Da V8 Laser Firing VY Processing Peak Cleanup Ml Monoisotopic Peak Picker VILE Peak Filtering VISA Mascot BMY Output v gt Data Storage Save Method Load Method Figure 15 15 Method Editor Mascot Parent Search Method Editor 179 180 Chapter 15 Automated operation Method Editor default mtd Sample Method ZIX Auto Quality Mascot MS MS Search ve Laser Firing WEY Processing Search Title FO VILE Calibration EZ Peak Cleanup Database MSDB v Decoy E vi Monoisotopic Peak Picker VEX Peak Filtering Report Top Hits 20 v Overview z vS Mascot OD Taxonomy All entries J Applications amp Compare Sequence Enzyme Trypsin x Missed Cleavages J v Oligo Analysis My Output Fixed Acetyl K p Modifications
152. 0 my TL Threshold response 1 000 x i Apply to Tiles Lull Lej Apply to Samples E Close Apply E Advanced 3 ie Poisson Figure 16 9 Setting a threshold on the Peak Cleanup window To set a threshold place a cursor on the spectrum display with the cross hair at the position where the threshold is to be set Using a Full cursor setting is helpful in this instance see Cursor width on page 361 Press the t4 button adjacent to Threshold on the Peak Cleanup window This will get the threshold level from the last moved cursor position Figure 16 10 Select the AY button to detect the peak area down to the baseline Peak detection 249 250 Chapter 16 Cleaning up data Peak detection Select the JEN button to detect peak area between the limits of the peaks Select the amp mira button to switch off the adaptive threshold feature Select the ly button to switch on the adaptive threshold feature lint 100 7 mV sum 744 mV Shots reo Smooth AV 12 7 Before setting threshold 804 704 604 504 ae Wide cursor 304 204 4467 07 m 104 o 4000 5000 5000 7000 8000 Mass Charge int 100 7 mV sum 744 mV Shots 1 100 Smooth Av 12 003 After setting ci nf threshold 804 704 604 504 404 304 204 104 o 4000 i 5000 5000 7000 8000 Mass Charge Figure 16 10 Setting a threshold
153. 00 00 2550 00 Ba Barium 56 137 32689 137 90523 7 725 00 1640 00 Be Beryllium 4 9 01218 9 01218 1 1278 00 2970 00 Bi Bismuth 83 208 98037 208 98037 1 271 30 1560 00 Bk Berkelium 97 247 07030 247 07030 1 0 00 0 00 Br Bromine 36 79 90353 78 91834 2 7 20 58 78 c Carbon 6 12 01104 12 00000 2 3550 00 4824 00 Ca Calcium 20 40 07802 39 96259 6 839 00 1484 00 Cd Cadmium 48 112 41155 113 90336 8 320 90 765 00 xl Figure 37 3 Element symbol property page Accessing the database 565 Chapter 37 Element database For the Periodic table property page the standard table of elements is shown as in Figure 37 2 An individual element can be selected using the mouse SELECT button to display the isotopes for that element and their abundance in the lower table Editing the Element Database The Element Database has been constructed using information obtained from compiled tables in Rika nenpyo Chronological Scientific Tables National Astronomical Observatory Ed Maruzen Co Ltd Japan which reference earlier work by A H Wapstra and G Audi Nuclear Physics A432 1985 1 and IUPAC in Pure and Applied Chemistry 63 1991 991 It should not be necessary to modify these values and indeed should an error be made in editing the database the repercussions would be that all mass calculations would be in error Exercise extreme caution in editing the database and double check any changes made before saving To start the Element Database Editor sel
154. 0006 0 angio2_msms0006 0 angio2_msms0006 0 angio2_msms0006 0 angio2_msms0006 0 Sort Increasing mass z Processed Processed Processed Processed Processed Processed zj Click right Show selected labels mouse button Hide all labels Show all labels Delete selected labels Delete listed Delete listed between cursors Delete all labels New Annotation Load Include Save Properties ok Amy Cancel New This window is also available from the right mouse menu options on any graphic display Figure 5 16 Annotation sub windows Processing menu Chapter 5 Window and menu guides Sequence Calculator sub windows Accessed by Processing gt Sequence Calculator this is described in Sequence Calculator on page 601 Sequence Calculator Lolk File Edit View Settings 4 gt Synctrorse Name Untitled Masses RESM E N terminus Hydrogen H z Moncisotopic 7 p Crterminus Hydroxy H 0 T Fragmentation Singly charged gt Cation Proton H z Digest amp Generate Peak Markers SBE Peak Marker Tolerance Settings Tolerance 0 8 4 Da Apply Tolerance V Load Sequence Database Shimadzu Biotech Launchpad Peptides angiotensin 2 ksq Select Keywords Sequence Mass range 1 1700 daltons Oo o 1047 20 Angiotensin 2 Lis
155. 04 Items in blue have no display contents S Intensity Mapping Contents x jw Instrument Record Listing x Dataset Trace Sample T tissue0001 x mje ieee Percentage intensity i HEELS Intensity values TIC Summasses Profle Max Figure 5 23 Display contents windows b Display contents windows 57 58 Chapter 5 Window and menu guides Display contents windows Chapter 6 Configuring Launchpad Chapter 6 Configuring Launchpad 60 Chapter 6 Configuring Launchpad Environment Configuration Editor Changing Launchpad file locations The data created by the Axima instrument is stored in specific folders on the PC The reference files and calibration files needed for data processing are also kept in specific folders the locations of which can be redefined by the user For example all users may wish to keep reference files in the same location to save users duplicating the reference files In order to specify where files are kept the Environment Configuration Editor is used The software installation procedure will create a set of default paths relative to the fixed hard drive C You can change the path Environment Configuration Editor Chapter 6 Configuring Launchpad 1 Access the editor from the Windows taskbar Start gt Programs gt Shimadzu Biotech Launchpad gt Programs and starting the config_ environment exe appl
156. 1 fa Programs Xe Favorites gt m a an Accessories gt HIRE IT Administrative Tools gt vi Settings MGi Shimadzu Biotech Batch Processor om IT Quadralay gt wi Release Notes FA p Search bs ffi i p IT RoboHelp Office User Guide om SmartDraw Help and Support a I Soundmax JE Run I Startup I Symantec Client Security 2 Log Off bill IE Volo View Express gt c z Turn Off Computer Start Figure 34 1 Starting the Batch Processor Using the Batch processor 539 Chapter 34 Batch processor XML export The Batch Processor window appears Batch Processor OF x Processing Type ASCII Converter Options Processing Settings Source Files Add Remove Clear Destination Folder Same as source Specific folder BROWSE Process Files Figure 34 2 Batch Processor window 2 At the Processing Type field from the drop down list select the required export format mzData Converter or mzXML Converter 3 If you selected mzXML Converter a select the Options button and select the required radio button from the mzXML Settings dialogue box mzXML Settings Eg Extract raw data Extract processed data Extract peak data Cancel Figure 34 3 mzXML Settings window b Select the OK button 540 Using the Batch processor Chapter 34 Batch proces
157. 10 91 6 77 1020 19 0 49 0 09 0 04 2915 50 30 13 1022 84 0 21 0 04 0 02 2746 17 16 94 1026 10 0 08 0 02 0 01 2924 02 6 80 1027 74 0 34 0 06 0 04 2861 46 29 21 1030 83 0 45 0 08 0 03 2694 56 22 20 1031 77 0 06 0 01 0 01 2865 70 6 91 1032 36 0 07 0 01 0 01 2901 55 10 65 1033 85 0 07 0 01 0 01 2943 74 10 76 1035 45 0 57 0 10 0 04 2833 03 28 63 1037 36 0 20 0 04 0 02 2921 49 18 58 1038 09 0 07 0 01 0 01 2941 01 6 82 1041 56 0 07 0 01 0 01 2802 80 6 93 1042 95 0 20 0 04 0 02 2698 38 14 63 1047 46 10 14 1 82 0 49 2405 37 381 78 1048 82 0 68 0 12 0 16 2287 29 126 17 1053 61 0 07 0 01 0 01 2963 01 6 78 1054 55 0 12 0 02 0 01 2973 21 10 71 1055 37 0 07 0 01 0 01 2992 31 6 87 For Help press F1 Figure 17 8 Example of a Mass list display Setting Maximum listed peaks restricts the report to the specified number of most intense peaks in the mass range of the report e g set Maximum listed peaks to 5 to see only the five most intense peaks in the selected mass range Small peaks can be filtered from the report by setting a Minimum peak apex Enter a value in millivolts Peaks below this height are not listed The minimum apex can also be set by placing a cursor at the appropriate height on a processed data display and pressing the cursor button t4 Certain types of processing flag peaks as significant e g polymer analysis flags all of the peaks which are part of a polymer sequence as significant These peaks are displayed in a diffe
158. 10 times to indicate dimer trimer and other multiple mass fragments These additional cursors will follow track the movement of the normal cursors on the display The example in Figure 20 16 shows cursors for up to 5 times the mass under the normal cursor Wherever the selected range cursor moves to the optional cursors will follow allowing easy location of multiply charged or multi mass fragments Figure 20 16 Chapter 20 Managing Data Displays Display Options BEI General Graphs Graph Text Text Report Cursors Peak Labels Type Charge Mass up to mz a Charge up Width None Normal Display Window Height Graph Display window Join with Off Line lon Gate Colours T All displays angio2_msms0006 MALDI MS O x File Edit view Instrument Automation Processing Help S bio EES 2j E x x Display Spectrum v Profiles fi Masses 1 1040 KA Data angio2_msms0006 G7 c 4 Mar 2005 14 37 Cal tof 4 Mar 2005 14 37 CID of 1046 51 Shimadzu Biotech Kompact MALDI 6 V2 7 0 Beta 1 Mode reflectron_ms_ms Power 73 Gate 1035 00 1060 00 P Ext 1050 bin 53 lnt 11 m sum 596 mY Profiles 1 54 Smooth Av 20 109 96 Normal cursor 2x mass Ko a 255 23 756 51 931 65 343 26 619 46 784 38 933 16 10 shh 208 23 394 23 saliai ragg5 WA 2354 344 50 psbg 908 47 i MUN hls nit Pa Mbadala a Fda Waapas et 100 200 300 400 600 700 800 900 Mass Charge
159. 12050 Cannot truncate sequence database file The file cannot be opened and truncated check that the file is a valid file and is non zero in length Error 12070 Unable to send email message The Windows event log may contain more specific descriptions of the errors encountered The system is unable to establish an email connection Check that you have a MAPI compliant email package installed for example MS Outlook Error 13000 Unable to load periodic table database Please correct Check that the file periodic_table data exists in the selected databases directory Error 14000 Unable to open temporary file for log window printing Error 14010 Unable to write header line to temporary file log window printing Check that the TEMP and TMP lines are set in autoexec bat check that a valid TEMP directory exists Error messages 653 Chapter 44 Summary of error messages Error 16000 Error 16010 Error 16020 Error 16030 Error 16040 Error 16050 Error 16060 Error 16070 Error 16080 Error 16090 Error 16100 Error 16110 Error 16120 Error 16130 Error 16140 Read failed for element mass value Read failed for element abundance value Read failed for element specific gravity value Read failed for element melting point value Read failed for element boiling point value Read failed for element conductivity value Read failed for element symbol value Read failed for element full name value
160. 147 18 2 176 204 3 233 261 4 380 408 5 493 521 6 649 677 Figure 42 26 Example of a mass spectral fragmentation report It can be seen from the example that PSD fragmentation produces six fragments for each fragment type A 17 and B 17 counting from the N terminus N C terminus terminus AA AG AA 630 Introduction Chapter 42 Sequence Calculator Applying peptide PSD fragments as peak markers Ensure that the Peak Markers button is selected View gt Options gt General tab The application of peak markers was discussed in Adding text annotation on page 387 To apply the PSD fragments generated by the Sequence Calculator as fragment peak markers press the Generate Peak Markers button on the Sequence Calculator window This will display the Generate Peak Markers window as shown in Figure 42 27 below amp Generate Peak Markers x Peak Marker Tolerance Settings Tolerance 0 8 4 Da Apply Tolerance IV Figure 42 27 Generate Peak Markers window When applying peak markers a Tolerance can be specified that marks only those peaks in the set of fragments generated that match existing fragments within this tolerance window Select the Apply Tolerance box to apply this tolerance If this box is left unselected all peak markers will be applied to the full spectra Press the Generate button to apply the settings This will generate a set of peak markers as shown in the Anno
161. 1TA plain de2961ta plt DE4555TA 384x2800 96 de4555ta plt Adapt ion 4x48 quickmass to 484r00 plt slides 4x48 fleximass to 483r00 plt ABI plates abix2 plt MassTech masstechx2 plt plates Plate file to use Other plate files may exist that include pre defined positions for calibrants although the Sample plate does not have wells for these For example plate file 384x2800 06 de1580ta pit includes six calibrant positions Sample plates and plt files Chapter 13 Preparation for data collection Before beginning to acquire data it is necessary that the sample plate is seated on the stage in a manner such that the position of the plate and all the wells on it are accurately known by the instrument The Acquisition Slide window is the starting point for the three possible stages in this process 4 Acquisition BEI Firing Exp Tech Auto Quality Storage Slide Raster Tuning Sample plate File Change Plate Carrier Edit sample plate Align plate Load Align Sample Plate 2 1X v Stage Control f eT 39 O my Cancel seconds Alignment References 1 JS Stage Bet ID Platex Platev Stagex Stage Al 72 250 114 250 19420 000 31703 000 A24 72 250 10 750 19497 000 5779 000 P24 4 750 10 750 2571 000 5813 000 Ready 17170 29453 Figure 13 11 Align Sample Plate window 1 Initialise stage This step is performed only when the stage is suspected of having errors and i
162. 2 15 Creating links within sequences The Loss entry on the Link Sequences window specifies the elemental formula of the group which is lost when the cross link is formed Usually in most cases this will be hydrogen H but it can be defined to be any species whatsoever The colour of the link indicates the sequence to which it is linked and the number specifies the number of the unit in the sequence In the next section you will see that any number of sequences can be displayed in different viewing panels The colour of the highlight Introduction Chapter 42 Sequence Calculator border surrounding the viewing panel can be user defined The link colour is the same as the colour of the viewing panel containing the sequence to which it is linked Using multiple viewing panels Up to ten viewing panels can be displayed simultaneously within the Sequence Calculator window To create a new panel select Insert panel from the Edit menu Figure 42 16 Sequence Calculator Ol x File Edit View Settings Gut Gh Grph pe Paste Ctrl Find and Replace Ctrl F Provecting arounsy GFF Links Ctrl L Insert panel Delete panel Clear markers Delete sequence Figure 42 16 Inserting a new panel A new panel will be inserted in the Sequence Calculator window Figure 42 17 Sequence Calculator Oy xi File Edit View Settings E PPPPPPPPPPPPPPR 5 10 15 e4 gt gt gt I Synchronis
163. 2 7 0 Bets 1 Mode refi Wint 6 7 mVVisume 955 mV Profiles 1 142 Smooth Arf Wint 1 5 mV sum 220 mV Profiles 1 142 Smooth Av 20 1297 16 100 h 1534 33 o coal eaii a a 13236 123 130 1302 1H TIC Mass Charge E a a ee Figure 20 5 Inserting a new column into the displays Multiple displays 343 344 Chapter 20 Managing Data Displays Displays can only be added as a complete row or column such that inserting above a row of two displays will add two new displays above the original ones as shown in Figure 20 6 When inserting a new column by default the newly inserted column will be half the width of the selected display and a newly inserted row will be half the height of the selected display These default sizes can be easily adjusted after the new displays have been created Shienedzu Biotech Kornpact MALDI6 V2 7 0 Beta 1 Mode reld Shimadzu Biotech Kompact MALDI 6 V2 7 0 Beta 1 Mode refi a Aint 6 7 mVisum 955 mV Profiles 1 142 Smooth Aw WF Wint 1 5 mV sum 220 mV Profiles 1 142 Smooth Aw 20 153433 1297 16 A 200 2500 T 16 ie 130 tao tanallel Git Mass Charge Mass Charge Cop z 46690005 MALDI MS pep m Da St You Format dennon Posen ip COLCCE r CECI E Jne Maes e i Pra Pole a jeje z a a Se rte a a Figure 20 6 Inserting a new row into the displays Deleting displays The Delete option on the Display me
164. 28 Sep 2005 13 34 CID of 1533 66 Data p14r_msms000413 c 28 Sep 2005 13 Shimadzu Biotech Axima ToF 2 7 0 20051223 Mode reflectron Power 62 Gate 1515 00 1555 00 Shimadzu Biotech Axima ToF 2 7 0 2005122 lnt 15 m surm 349 mv Profiles 1 23 Smooth Gauss 20 Baseline 50 lnt 167 miv sum 3842 mv Profiles 1 195 0470 r323 1633 8580 r2193 100 100 1341 2362 1545 1534 8621 12739 90 90 Wi 80 804 70 704 60 60 so 6 0995 r 179 50 a 9512862 313 40 1342 6 404 2 2003 r544 1145 5434 r465 30 30 j 1146 8715 1343 20 536187 13 r364 10 at ll a MAHI daital a ee 200 400 600 800 1000 1200 1400 1530 1535 1540 Mass Charge Mass Charge Table 28 2 Actual masses P14R MS MS spectrum with the fragment calibration applied Data p14r_msms0004 13 c 28 Sep 2005 13 35 Cal fc_test 28 Sep 2005 13 34 CID Data p14r_msms0004 3 c 28 Sep 2005 13 Shimadzu Biotech Axima ToF 2 7 0 20051223 Mode reflectron Power 62 Gate 1515 00 1555 00 Shimadzu Biotech Axima ToF 2 7 0 20051 22 lnt 15 m sum 349 mY Profiles 1 23 Smooth Gauss 20 Baseline 50 lnt 167 m sure 3842 mv Profiles 1 525 7 B00 rB96 1 1533 8580 r27 64 100 100 E 1406 67 1 18 1534 52 1 12739 90 90 80 80 70 70 60 60 a 2 1536 8665 r2652 40 1407 6386 r4t 40 E 1274 1723 r751 30 9 30 20 10 i 0 1000 1200 1400 1530 1535 1540 Mass Charge Mass Charge Axima Performance Chapter 28 Frag
165. 39 Searching for molecular weight matches The calculations can be performed using either Average or Most abundant or Monoisotopic masses see Instrument Calibration on page 459 for more details on these terms Having made all of the selections press Search to begin the search for matches The search will stop when either no more combinations are possible or a thousand 1000 matches have been found This technique is really only viable at low molecular weight because the number of elemental combinations increases tremendously with an increase in molecular weight For this reason the chances of finding a good match at high molecular weight is considerably reduced In the example shown in Figure 39 2 a molecular weight match is being sought for a fragment of mass 243 2 2Da The results of the search are listed in the bottom panel of the window Figure 39 3 The matches are listed in increasing error difference order the closest match being first in the list next previous match number of this match total number of matches found limit 1000 molecular weight of ae the match _ Match 1 2 of 20 Mass 1000 0324 Daltons Error 0 0324 Daltons 32 39 ppm Formula difference in mass between match and specified molecular weight list of elements species in the match Figure 39 3 Matches displayed in the Search window In the example given one match was found being a molecule comprising Phe Pro of mole
166. 4 Averaged Average 480 mV sum 25926 m Profiles 1 54 Smooth Av 20 1046 5 Process 1047 5 1048 5 7148 m Profiles 1 54 Threshold 25 Centroid 1046 5 1048 8 1049 6 Figure 12 3 Example of the four spectrum trace types 110 Displaying spectrum Chapter 12 Introduction to displaying data An example of Stack and Overlay displays is shown below 1046 1047 1048 1049 1050 1046 1047 1048 1049 1050 Stack Overlay Figure 12 4 Example of Stack and Overlay traces Displaying spectrum Chapter 12 Introduction to displaying data Multiple sample datasets Where more than one sample is available for a dataset scroll through the sample using the up down icon E Spectrum Contents Dataset Trace Sample Process 1 angl1__0001 Scroll dataset Traces Profile y Process Peaks View Stack Overlay Set 15 10 p n Select to scroll through samples Select to scroll through datasets The spectrum display updates without the need to select the Apply button Similarly the dataset scroller is available to allow at a click automatic updating of the datasets displayed on a spectrum report Selecting the button will if possible deselect the highest numbered selected and loaded dataset and select instead the nearest lower loaded but deselected dataset Selecting the button has the reverse effect if possible the lowest numbered selected and loaded dataset is deselected
167. 6 Configuring Launchpad Mascot Setup Mascot Setup The Mascot Setup property page Figure 6 2 contains settings required to implement the Mascot Search facility in the Launchpad Method Editor see Mascot Searching on page 178 There are two methods of using Mascot Remotely you use the internet to access the Mascot search engine at the Matrix Science web site e Locally your organisation has a Mascot server connected to your LAN This method is required for automated experiments where Mascot is being interrogated frequently To perform a Mascot search paths to the Mascot server need to be specified Usually these parameters are set by the service engineer during installation Environment Configuration Editor Environment Variables Mascot Setup Email address me kratoscou 0 Search URL http www matrixscience comjegifnph mascat exe 1 FTP results server Po FTP results location mascot data O FTP intermediate processing location mascot_tempresuts 00 FTP config location mascot config 0 J The server requires a username and password when connecting Print Settings Reset Apply Help Figure 6 2 Environment Configuration Editor Mascot Setup Chapter 6 Configuring Launchpad The figure above shows typical settings for remote Mascot setup For inter working with a local Mascot Server it is essential that the parameters set in this window mimic those that are setup on the Masc
168. 8 11 31 81 4 769792 v 100 4 78 11 31 B1 5 769792 v 100 4 78 11 31 81 6 769792 v 100 4 78 11 31 B1 Save Prescan Data Auto Quality Data Profile Laser Power Peak Intensity m Resolution Smoothing Off S N 1 Consecutive Fails XY Position x 65 v 2000 9228 X 62 x 4 78 11 31 B1 x 66 v 2000 v 9228 x 62 x 2 4 78 11 31 81 x 65 v 2000 v 9228 X 62 x 1 4 78 11 31 B1 x 66 v 2000 v 9228 X 62 x 2 4 78 11 31 B1 x 65 v 2000 Z 9228 x 62 xXx 1 4 78 11 31 B1 Save Auto Quality Data Save All Data Cancel Figure 14 12 Auto Quality View Data window Automated data quality filtering 156 Chapter 14 Collecting data from a sample Data is presented on a per profile basis including failed attempts and the figure given for resolution ignores any smoothing applied in Peak Processing A green tick or a red cross is shown in appropriate categories to indicate success or failure Failed attempts are where the profile did not meet the Auto Quality criteria for example resolution signal to noise etc You can store all relevant data and subsequently view it in a pop up dialogue box by selection of the appropriate check boxes Additional buttons are available to save the data to a delimited text file A separate check box gives you the option of overwriting or adding to any data previously stored in the data view for a given Auto Quality run If Prescan is selected for a series of points in a raster each po
169. 85 84 17 68 U 1061 42 1 62 1 11 135 99 35 08 4 12 4 64 708 46 55 13 U For Help press F1 l Nu f Figure 20 4 Multiple displays in a window 342 Multiple displays Chapter 20 Managing Data Displays Inserting new displays The display with the thick highlighted outline is called the selected display A display is selected by a single click of the mouse SELECT button and the display outline will be highlighted to show that the display was selected Where there is only one display in the window the outline is not shown The Insert option on the Display menu presents the Insert menu see Figure 20 5 This menu allows other displays to be inserted as either a column or row adjacent to the selected display Figure 20 5 shows the process of inserting displays using the Insert menu pep _mix_ 246600005 MALDI MS Ele Cdk Yew ratrumere Automation Processing tiep oki fle 2 amp ne ep Ossie Secon Poe i102 Maresfrovzme E Data pep_mix_246560005 I10 c 10 Feb 2005 10 06 Cal tof 10 Feb 2005 9 02 Shenadzu Biotech Kornpact MALDI 6 V2 7 0 Bets 1 Mode reflectron_ms Power 45 Blanked P Ext 2460 bin 91 int 6 7 mV sum 955 mV Profiles 1 142 Smooth Av 20 Baseline 80 153433 1600 1800 2000 2200 Mass Charge Cop Paste Delete Annotate Annotation Properties Tags Peak labelling Stenedzu Biotech Kornpact MALDI 6 V2 7 0 Beta 1 Mode refid Stenadzu Biotech Kornpact MALDI 6 V
170. AXIMA Launchpad 2 9 User Guide a i on i OBLOTECH peer yw 2 9 1 LAUNCHPAD E eee SHIMADZU BIOTECH bringing analysis to life Contents Contents osis a a O E 1 ntr d cti H eisicciiienciisncuis snwnenenciauanae naa a aaa 7 About this User guide cceeeeeeee cece eee teeta eee eee nena eta eeenaes 8 Printed QuideS i avsiecaveh sinner nni n commandline sabre creas wkedenedns 11 Getting help cv cciceccee ceva cde ce ethe conde s nRa EEE chats eeka gerne 12 Safety WarningS siisiissitissises riceeaseandaseenireaniaet ciasensiadansedeedadeeniies 13 Health and safety precautions c cece cece eee eee eee teat eens 14 Safety statements and warningS cceceeeeeeee eee eee eee ee eaten 16 Sample FECOFS cccceceeeceece cee eeee eee eeeeaeeseeeaeeeeeeneeenteneenens 19 Getting Started cccccceeeeeeeeneeeeeeeeeeeeeeeeeeeaeeoeaeeaeaseaeaeeaeaseaeeenseeans 23 INtrOCUCTION 0 20 cece eter ener ne eee ee ete ne annern eee 24 Introduction to the MALDI MS software ccceeeeeeeeee ees 28 The Launchpad cc cceee cece e eee eee eee eee eens ene een eee a tetas 30 Status of the AximMa c isccseeceeetee ted nnn eareee eee eten hea eee eke 32 Additional Quid c sccccseeeeeeeceeeeeeeeueeeeueeeeuaeeaeaeeeuaeeauaeeaeeauenens 33 PMEPOGUCEION E Ae laws dave teaeiane T 34 Window and menu guides cceeeeeeeeeeeeeeeeeeeeeeeeeeoeeseoueeeaeaeeaueans 35 MALDI MS
171. Acetyl N term v Print Piin Acetyl Protein N term DO Export W Report X 1 lon Finder Variable Acetyl K MEI MS MS Amidated C term Modifications Acetyl N term Acetyl Protein N term 3 MY Acquisition Amidated C term SE Peak Selection Vv Ea Raster Max Peaks 20 Protein Mass kDa 0 MH Laser Fiing Mass Types Monoisotopic VI Processing EK Peak Cleanup Ml Monoisotopic Peak Picker Peptide Tol 1 Da x MS MS Tol 05 Da x E VEA Peak Filtering RR Eh My Output C Average Instrument Default X ICAT Peptide Charge 74 X V3 Print Export Low Mass Limit 15 of Precursor Segments 1 I Report 1 lon Finder High Mass Limit 95 of Precursor Data Storage Save Method Load Method Method Editor Figure 15 16 Method Editor Mascot MS MS Search Most parameters of the Mascot interface are common to both a Parent and PSD Mascot search however they do differ slightly Search Title Type in the title of the search that will appear in the results page Database Select the relevant sequence database to be searched For a Parent mass search the dbEST database is not available since the entries are short sequences and not complete proteins For an automatic decoy database search select the Decoy checkbox Taxonomy Select a species or group of species in which to perform the search against Chapter 15 Automated operation Enzyme Select the r
172. Argon Other Block lt MS2 gt lt Acquisition gt lt PeakSelection gt lt MaxPeaks unsigned gt lt MinMass unsigned integer Da gt lt MinIntensity unsigned integer gt 0 100 percent ASCII Text Method file format 217 218 Chapter 15 Automated operation lt SequenceOrder enumeration gt IncreasingI ntensity ncreasingMass DecreasingI ntensity DecreasingMass lt MaxParentMass unsigned integer Da gt Block lt MSI gt lt Processing gt lt Calibration gt lt Reference float mass string formula gt lt Tolerance unsigned integer gt lt ToleranceUnits enumeration gt Da mDa ppt ppm lt FitThruZero enumeration gt true false lt OutputFile string gt must have write permissions Multiple Reference values are allowed Block lt MSx gt lt Processing gt lt PeakCleanup gt lt SmoothMethod enumeration gt Off Average Gaussian SavitskyGolay lt SmoothWidth unsigned integer gt lt BaselineSubtract enumeration gt true false lt BaselineWidth unsigned integer gt gt 0 lt PeakMethod enumeration gt ThresholdApex ThresholdCentroid GradientCentroid ThresholdC entroid25 GradientCentroid25 lt PeakWidth unsigned integer gt gt 0 lt PeakRejection unsigned integer gt lt PeakThreshold float mv gt lt PeakArea enumeration gt ToBaseline ToLimits lt Average enumeration gt All Tagged Block lt MSx gt lt Processing gt lt MonolsotopicPic
173. Calibration Bik Calibrant references r Calibration files lt Untitled gt J11 List references Reference editor Compounds Name tof List Save Formula Abundance Cursor mass Time Load named calibration x Load 190 05 H 1 E Calibration Fragment Correction _ x Apparent mass Actual mass Formula Fit error Da Reference filename P14R_TOF2 frag_ret Save it through zero Sav Apparent mass 458 8448 m Insert Delete Clear list tt Actual mass 195 1100 Formula Tol 500 mDa 7 F Combined Cal Calibration parent mass 1533 8580 fal Average vy 71000 Fit fragment calibration a 10 The fragment reference file P14R_TOF2 can be loaded from the setup window Alternatively if no reference file is available type in the actual and apparent masses given in table above 11 Ensure that the parent mass is set to 1533 86Da actual and apparent 12 For each fragment peak in the list a select the peak by double clicking on the entry in the list b enter the apparent mass the mass without the fragment calibration applied of the fragment peak in the P14R ms ms spectrum in the apparent mass box c press insert to enter the peak into the list d if the peak was from the list the message box peak within 0 5Da will appear asking if you want it replaced press OK to accept the new apparent mass value 13 Save the fragm
174. Chapter 24 Choosing user defined colour schemes Changing chromatogram trace colours To change the colours defined for chromatogram displays select the Chromatogram Colour Editor shown in Figure 24 4 Colour Editor Chromatogram BEI Colour sets i Load Load defaults Apply Figure 24 4 Colour Editor for chromatogram traces There are five colour choices on a chromatogram display Y axis backdrop X axis backdrop Mesh line colour Mesh Solid colour and the Front face colour Figure 24 5 shows the effect which the colour box choices have on the three dimensional chromatogram displays Ss ag Mesh colour Solid colour 600 1000 1600 Profile Front colour Figure 24 5 Example of chromatogram colour options Changing chromatogram trace colours Chapter 24 Choosing user defined colour schemes Having made all of the required selections press the Apply button to apply the selections to all of the displays This will apply the new colour scheme to the displays but the colour scheme modifications will not be saved in any way they are only temporary To save the user defined colour scheme so that it will be made the default colour scheme and loaded automatically when the MALDI MS software is loaded next time press Save If the colours have been modified for a temporary change and the previous colour scheme is required to be reinstated press Load and the previously saved scheme
175. Chapter 43 Listing of the template itn file CYSTEINE_ MENU lt Cysteine page name gt Unmodified lt Val 1 gt Acrylamide lt Val 2 gt lodoacetamide lt Val 3 gt 4 vinyl pyridine lt Val 4 gt Aminoethyl lt Val 5 gt Benzyl lt vVal 6 gt Other lt Val 7 gt Methionine oxidised METH_OXIDISED_CHECK lt Meth oxidised page name gt CHECKED lt Val 1 gt NOT_CHECKED lt Val 2 gt Number of missed cleavages allowed MISSED_CLEAVAGES_TEXT lt Page name gt Sequence Molecular Weight of whole protein SEQ_MOL_WEIGHT_TEXT Page name gt Search proteins of Seq Molecular Weight this percentage value FILTER_TEXT Page name gt Weighting between 0 and 1 0 given to partially cleaved peptide fragments PFACTOR_TEXT Page name gt Mass List MASS _LIST_TEXTAREA lt Mass List page name gt Number of peptides required for protein match NO_FOR_MATCH_EDIT lt No for match page name gt Number of Matches to show MATCHES _TO_SHOW_EDIT lt No of matches gt Chapter 44 Summary of error mess Chapter 44 Summary of error messages ages 647 648 Chapter 44 Summary of error messages Introduction Introduction This section lists the error messages which may be encountered while using the MALDI MS software A fuller description of the message is given and where possible a suggested reason for the error occu
176. D on 909 02 2000 Data Acquisition CID on 1618 91 Mass Charge 230 Using Auto Experiment Results viewer Chapter 15 Automated operation Mascot display The following image shows a Mascot result of the selected data displayed in a left hand column 4 0023_0001 318 1881_0001 AE0023_0001 H18 969_0001 MALDI MS File Edit View Instrument Automation Processing Help is ej ov baec Mascot Search Results User Administrator Email Mascot Search Search title Investigation MS data file Automatically uploaded data Data File Database Sprot sprot 208005 sequenc Timest amp 16 Aug 2006 at 13 55 47 GMT C Program Files Shimadzu Biotech Launchpad Data Cus Significant hits ALBU BOVIN P02769 Serum ir ALBU FELCA P49064 Serum C Program Files Shimadzu Biotech Launchpad Data Cusl Results Start End 16 08 2006 14 46 48 16 08 2006 14 48 33 C Program Files Shimadzu Biotech Launchpad Data Cusl Shiv j q z Prob ability Based Mowse Score C Program Files Shimadzu Biotech Launchpad Data Cus E C Program Files Shimadzu Biotech Launchpad Data Cus Tons score is 10 Log P where P is the probability that the observed match is a random event Individual ions scores gt 36 indicate identity or extensive homology p lt 0 05 Click to view Mascot peptide summary results Results For Help press F1 Multiple tiles Refer to the Getting
177. DOS window from the Start menu and type ascii2plate myinputwell txt fourwell plt This will by default give the plate the description Sample spots and the plate dimensions width 77 0 mm and height 124 0 mm and an X and Y offset of 0 0 mm To define different overall plate parameters optional command line arguments can be supplied i e d description wwidth hheight xoffset yoffset So typing and entering the single line ascii2plate d 4 spot well w50 0 h120 0 x4 0 y6 0 myinputwell txt fourwell plit would create a plate file fourwell plt with the same well descriptions from myinputwell txt as before but with width and height 50 0 and 120 0 mm respectively X and Y offsets of 4 0 and 6 0 mm respectively and the descriptive text 4 spot well Sample plates and plt files 127 128 Chapter 13 Preparation for data collection Plate Alignment for Axima instruments Kratos supplies a range of standard plate types and corresponding plt files are shipped on the release CD These are Sample plate DE1271TA 384x2000 00 de1271ta plt DE1487TA 96x4700 00 de1487ta plt DE1579TA 384x3400 00 de1579ta plt DE1580TA 384x2800 00 de1580ta plt DE1583TA 96x3400 00 del1583ta plt DE1798TA plain de1798ta plt DE2110TA plain de2110ta plt DE2111TA 96x4700 00 de2111ta pit DE2112TA 96x3400 00 de2112ta pit DE2113TA 384x2000 00 de2113ta pit DE2114TA 384x3400 00 de2114ta pit DE2115TA 384x2800 00 de2115ta pit DE296
178. E Melting point Boiling point Contents Total number of elements in the database Element index in the database The name of the element The elemental symbol for the element The atomic number of the element The minimum double bond equivalence not used The maximum double bond equivalence not used The melting point of the pure element in Celsius The boiling point of the pure element in Celsius Accessing the database 567 568 Chapter 37 Element database Table 37 1 Element Database fields Field Contents Specific The specific gravity of the element gravity Conductivity The electrical conductivity of the element Up to 10 isotopes can be entered in any order the isotopes will be automatically sorted upon pressing the Sort button The isotopes can be sorted in Mass or Abundance order Press Save to save the database or Reset to restore the previously saved database The file which will be created is periodic_table data and it will be written to the location specified by the Databases path in the Configuration Editor see Environment Configuration Editor on page 60 It is worth making a backup copy of this file prior to editing as this file is crucial to the calculations performed by the MALDI MS software Accessing the database Chapter 38 Creating a compound database Chapter 38 Creating a compound database 569 Chapter 38 Creating a compound database Introduction More of
179. E IH Raster viv X Auto Quality v3 Laser Firing J Processing MIL Calibration EX Peak Cleanup II E Monoisotopic Peak Picker Node always MALS Peak Fitering selected regardless MS Mascot of method CY Applications Compare Sequence Oligo Analysis My Output Wi Print CI Export I Report lon Finder K Node not in current method Figure 15 2 Tree Structure of a Method A checked box next to a node in the tree indicates parameters for this option will be used in the Method Nodes that require to be omitted from the current Method should be left unchecked To display the dialog for a node select left mouse click the text label of the required node Method Editor a S 168 Chapter 15 Automated operation Method Editor There are two main branches of the Method Editor For Axima instruments these are the Parent and MS MS branches If selected the Parent branch will perform acquisition of a parent ion mass spectra for a PMF experiment or regular auto run experiments If selected in conjunction with the parent branch the MS MS branch will perform MS MS acquisition processing on peaks from the parent spectra for a PMF experiment If selected independently standard MS MS spectra will be acquired For Axima QIT instruments the two main branches are MS mode data acquisition and MS acquisition The functionality is governed by the same rules as described above for Axima Confidence operation The curre
180. ESS gt Chapter 43 Listing of the template itn file Species TAXONOMY_MENU lt TAXONOMY page name gt ALL lt Val 1 gt Fungi lt Val 2 gt Viridiplantae Green Plants lt Val 3 gt Mammals lt Val 4 gt Homo Sapiens lt Val 5 gt Viruses lt Val 6 gt Bacteria lt Val 7 gt E Coli lt Val 8 gt Archaea lt Val 9 gt Eukaryota lt Val 10 gt Metazoa lt Val 11 gt Drosophila lt Val 12 gt Chordata lt Val 13 gt Other lt Val 14 gt Digest Enzyme ENZYME_MENU lt Enzyme page name gt CNBR lt Val 1 gt Trypsin lt Val 2 gt Arg C lt Val 3 gt Asp N lt Val 4 gt Lys C lt Val 5 gt V8 DE lt Val 6 gt V8 E lt Val 7 gt Chymotrypsin lt Val 8 gt Other lt Val 9 gt Mass Type protonated neutral MASS_TYPE_MENU lt Mass charge type gt Protonated MH lt Page Value 1 gt Neutral M lt Page Value 2 gt Monilsotopic masses MONOISOTOPIC_CHECK lt Monoisotopic page name gt CHECKED lt Val 1 gt NOT_CHECKED lt Val 2 gt Minimum mass MINIMUM_MASS EDIT lt Min Mass pagename gt Maximum mass MAXIMUM_MASS_ EDIT lt Max Mass page name gt Tolerance TOLERANCE EDIT lt Tolerance pagename gt Tolerance Units TOL_UNITS MENU lt Tolerance units page name gt Da lt Val 1 gt lt Val 2 gt ppm lt Val 3 gt mmu lt Val 4 gt Cysteine Modification eoeecee oa A on 646
181. Fragment lon mass Comment a N M lons formed by main chain b CO fragmentation with the c N M positive charge on the N N M N_ terminus H3 a 1 a H Addition of 1 or 2 hydrogen a 2 a H2 atoms b 1 b H b 2 b H2 a 17 a NH3 Loss of 17 Da Probably due b 17 b NH3 to loss of NH3 c 1 c H Loss of 1 or 2 hydrogen c 2 c H2 atoms d lons produced by side chain d fragmentation Chapter 42 Sequence Calculator Table 42 3 N terminus ionisation fragments Fragment lon mass m References for the above table Comment Internal immonium ions Formed by the loss of one of the side chains from the complete peptide fragment 1jJohnson R S Martin S A and Biemann K Collision Induced Fragmentation of M H lons of Peptides Side Chain Specific Sequence lons Int J Mass Spectrom lon Processes Vol 86 Pp 137 154 1988 2 Johnson R S and Biemann K Computer Program SEQPEP to Aid in the Interpretation of High energy Collision Tandem Mass Spectra of Peptides Biomed Environ Mass Spectrom Vol 18 Pp 945 957 1989 Table 42 4 C terminus ionisation fragments Fragment lon mass X C M C y O Z C M H gt C M NH x 1 X H X 2 x H2 y 1 y H y 2 y H2 z 1 z H Z 2 z H2 y 17 y NH3 V W w References for the above table Introduction Comment lons formed by main chain fragmentation with the positive charge on the C terminus Addition or loss
182. Guide Axima Confidence Getting Started Guide Axima Assurance Getting Started Guide Analysing Polymers Analysing Proteins using LC MALDI Analysing Imaging Experiments Analysing Oligonucleotide Experiments Customer Support Guide Release Notes License Agreement About MALDI MS The PDFs are designed for printing although you can view them through Adobe Acrobate Reader Introduction Chapter 5 Window and menu guides Chapter 5 Window and menu guides 36 Chapter 5 Window and menu guides MALDI MS base window The MALDI MS base window has seven menu items on it which display pop up menus The base window is described in Introduction to displaying data on page 107 4 angio2_msms0006 MALDI MS Eile Edit View Instrument Automation Processing Help Spectrum hd Ctrl 0 Ctrl Open Save Save As Open Auto Experiment Results Comments Parameters gt Print Print Report Print Preview Print Setup Export b Exit Acquisition Maintenance Paste Ctrley Delete Del Copy Window Copy Selected Tile Copy Entire Report v Toolbar v Status Bar Display Toolbar v Basic Parameters Display Contents Options Camera Instrument Status Database Viewer Tile Manager Events Logged Figure 5 1 MALDI MS base window QIT ToF Experiment a Help Topics User
183. Hydrogen H C terminus Hydroxy H O Sequence composition F G2 I L R3 Y Elemental composition M H C52 H85 N18 O11 Average mass M H 1138 37 Bull Breese 2360 00 Most abundant mass M H 1136 13 HPLC 38 50 Monoisotopic mass M H 1137 66 rypsin digest Average Masses M H N Position Mol Wt BB HPLC Elemental Formula Sequence 1 1 6 712 83 2290 0 45 7 C34 H50 N9 08 YGGFLR 2 7 7 175 21 690 0 3 0 C6 H15 N4 02 R 3 6 9 288 37 760 0 9 6 C12 H26 NS 03 IR Figure 42 25 Example of a theoretical Trypsin digest of Dynorphin From the example it can be seen that there are three digest fragments arising from cleavage at positions 1 6 7 and 8 9 in the original peptide chain positions are given relative to the N terminus The mass of the digest fragments produced and the amino acid composition are given for each fragment Figure 42 26 gives an example of the PSD fragmentation for the first enzyme digest product this fragmentation is for A 17 and B 17 ions Introduction 629 Chapter 42 Sequence Calculator I Dynorphin trypsin digest fragment number 1 L 6 YGGFLR 10 N terminus Hydrogen H C terminus Hydroxy HO Sequence composition F G2 L RY Elemental composition M H C34 H50 N9 08 average mass M H 712 83 Bull Breese 2290 00 Most abundant mass M H 712 67 HPLC 39 10 Monoisotopic mass M H 712 38 Singly charged Fragments Average Masses M H a 17 b 17 n 0 il 119
184. MS window click the Adjust Gate button Quick Gate Adjustment xi Mode Massrange mMid850 Polarity Positive x Parameters Point1 Point2 Set mass 1000 85 Da 2521 17 Da Actual mass 1001 63 Da 2519 08 Da Reset Results Gradient 1 0018913 Offset 2 674413 OK Cancel 2 Select the Mass range and Polarity fields to the required values Calibrating the ion gate 501 502 Chapter 29 lon gate calibration 3 Enter the Set mass and Actual mass actual mid point of the ion gate values for the lower Point 1 and upper Point 2 calibration masses 4 Click the Calculate button the calibration is calculated Repeat the above for any other modes and polarity 6 Click the OK button i Calibrating the ion gate Chapter 30 Chromatography Chapter 30 Chromatography 503 504 Chapter 30 Chromatography Introduction The Chromatography window provides tools for automatic peak detection in collected data over specified mass ranges with a variety of detection and processing options To use the Chromatography window select Chromatography from the Processing menu Figure 30 1 p14r_msms0004 MALDI MS Oy x File Edit View Instrument Automation Processing Help AE Jae a x Calibration Profiles 1 Masses 12 1040 1 gt Annotation Peak Processing Chromatography Polymers Sequence Calculator Reference Editor Se
185. Manual im Edit Mass List Insert Delete gt All masses bd wil Figure 5 14 Processing windows b Processing menu 49 50 Chapter 5 Window and menu guides Calibration Annotation Peak Processing Chromatography Polymers Analysis Sequence Calculator Reference Editor Select Peaks Manual Peak Assignment Internet Search Internet search Select the engine to be used for your search from the list below ss Search settings This section allows you to specify a webpage URL to be used when submitting searches Once selected you can choose the type of database you wish to use Search URL http www matrixscience com cgi nph mascot exe 1 Database Reporting Ba Use these options to configure the sections of the report which the server e mail addr creates for you You may also personalise your report with your name and ess Name l E mail address Report title zi cmn Figure 5 15 Processing windows c Annotation sub windows Accessed by Processing gt Annotation this is described in Annotation on page 382 Annotation SBE Hide selected labels Display labels list Trace All Traces X y0 19 02 b 17 1 99 10 i1 88 09 y1 166 20 b 17 2 255 28 i2 129 19 Tye E Z Dataset A1 datasets angio2_msms0006 0 angio2_msms
186. Manual Axima Performance Getting Started Guide Axima Confidence Getting Started Guide Axima Assurance Getting Started Guide Analysing Polymers Analysing Proteins using LC MALDI Analysing Imaging Experiments Analysing Oligonucleotide Experiments Customer Support Guide Release Notes License Agreement About MALDI MS Calibration Annotation Peak Processing Chromatography Polymers Analysis Sequence Calculator Reference Editor Select Peaks Manual Peak Assignment Internet Search Optional features and instrument specific items are in red MALDI MS base window Chapter 5 Window and menu guides File menu This menu controls all aspects of file handling selection of files opening and saving This is described in Loading and unloading data on page 73 D pte _rses00003 000001 nun piar masio Dpr4r_mams_po_gsso001 run EE bec ES Bee Data Fes nan ca Loki Osconms 5 ea j protein mbe Open Ctrl o Save Ctrl 5 Save As Open Auto Experiment Results Parameters bas a A Print Report Flevctbee Eparmere Rands Fies re z Cwen Print Preview E oaot 1 bango 2 eth theoreti aon p O O Oo Sgectwet Uae at erie ASCII Window as a MetaFile Selected Tile as a MetaFile mzXML File mzData File Intensity mapping as ASCII Ton Finder Biomap Lal
187. Peak Selection C Ea Raster 1 3 Laser Firing I Processing ER Peak Cleanup CIM Monoisotopic Peak Picker CIEE Peak Filtering 92 Mascot My Output Data Storage Get Manual Settings Save Method Load Method Figure 15 28 Method Editor Export Any exported data is automatically saved to a file in a similar manner to the current method filename in the default export path e g C Program Files Shimadzu Biotech Launchpad export default txt Intensities are exported as raw counts unless the Report Intensities as mV is selected Method Editor Report Chapter 15 Automated operation The Method Editor Report window provides functionality to produce a report file as a result of a Sequence match or the mass list of the current methods dataset To access the Report window select the Report label from the tree as shown in below Method Editor default mtd Sample Method Sample Method default mtd iy Parent El MY Acquisition M fa Raster VX Auto Quality 3 Laser Firing VE Processing VIL Calibration EEX Peak Cleanup MILE Monoisotopic Peak Picker VIER Peak Filtering vS Mascot SY Applications _ amp Compare Sequence Oligo Analysis v I Output V Print Vi Export a ie lon Finder MS MS vI Acquisition BE Peak Selection v Ea Raster M 3 Laser Firing vi Processing fa Peak Cleanup M TA Monoisotopic Peak Picker MEX Peak Filtering VS Mascot My Output X Data Storage SE P
188. S Search Archiver Enzymes SaL Elements Compounds Polymers Figure 3 7 The Launchpad window The Launchpad 31 32 Chapter 3 Getting started Status of the Axima The status of the Axima is shown in an icon on the bottom right hand side default position of your monitor lt QOeg 13 17 Axima status icon The colour of the icon indicates the status of the Axima Icon 0900 Table 3 2 Status icons Status Green instrument OK and in Operate mode Dark green instrument OK and in Standby mode Yellow instrument has warnings Red instrument has failed Double click the icon to display the Instrument Status window Status of the Axima Chapter 4 Additional guides Chapter 4 Additional guides Chapter 4 Additional guides Introduction Various manuals are supplied with your Axima e the appropriate Getting started guide for your Axima supplied as a book the Customer support guide which details various topics related to your Axima but not appropriate for this user guide This is supplied as a book with the Axima Application guide s these guides describe how to use optional features which you may or may not have They are supplied as books with the Axima You can access PDF versions of some of these guides from MALDI MS File Edit View Instrument Automation Processing Help 5 a D Help Topi wa Hae 2 S re l l feet Axima Performance Getting Started
189. Smoothing can be applied to the chromatograms by selecting the Smooth option on the chromatogram Display contents window The degree of smoothing applied is controlled by the smoothing Level option Increasing this value increases the smoothing factor Figure 17 16 shows an example of different smoothing levels applied to collected data The data has been processed to display the largest intensity in each profile 288 Displaying Chromatograms Chapter 17 Viewing the collected data lnt 100 99 mV mass 1000 to 15000 Unsmoothed ms a ner cS EE me Ze ik fz POLL PF TTT POLL TILA TILIA EEA ZZ LLLA E AO n m r pa r a no panara LTE A ARATE A PALL LL 500 1000 1500 2000 Profile lnt 100 35 mY mass 1000 to 15000 Smoothed 1 Smoothed Level 1 ROT ees a e a a a are ea A a A AOU LLL LLLA DA a a a s A LAA LA Z nara lt ma Ep PO os wae ome ETT AR ELE WEA LAT PLE FF O 2 LAT TAER A IX LZ as ZL Ete DLL BATT LPF Ze rar a gh zz 5 par y 500 1000 1500 2000 Profile lnt 100 14 mV mass 1000 to 15000 Smoothed 2 Smoothed Level 2 100 50 GA 500 1000 1500 2000 Profile Figure 17 16 Smoothing levels applied to chromatogram displays Displaying Chromatograms 289 290 Chapter 17 Viewing the collected data Displaying laboratory notes After collecting and
190. University of St Andrews 417 auto experiment MS_MS rex Double click within the node Ela pie So 8 For Help press F1 Using Auto Experiment Results viewer 225 Chapter 15 Automated operation To expand a node move the mouse pointer over the node and double click ff AE0023_0001 318 1881_0001 MALDI MS olx File Edit View Instrument Automation Processing Help sjal e 2 2 play auto experiment Resuts Profiles 1 16 Masses 1459 2426 E C Program Files Shimadzu Biotech Launchpad Data Customers University of St Andrews 417 auto experiment MS_MS rex Data Acquisition Data Acquisition CID on 968 52 Data Acquisition CID on 1251 74 Double click within the node Data Acquisition CID on 909 02 Data Acquisition CID on 1618 91 Data Acquisition CID on 925 04 Mascot Search Investigation For Help press F1 NUM Z To compress the tree node double click the node 226 Using Auto Experiment Results viewer Chapter 15 Automated operation You can continue to expand the node tree by double clicking within the new nodes to reveal further information Z AE0023_0001 318 1881_0001 MALDI MS olx File Edit View Instrument Automation Processing Help Sjaj Jae 2 4 display auto Experiment Results w Profiles 1 16 v Masses 1459 2426 KA C Program Files Shimadzu Biotech Launchpad Data Customers University of St Andrews 417 auto experiment MS_MS r
191. _acid Fer C10 H10 04 194 1873 194 0578 194 0579 Gentisic_acid Gen C H6 04 154 1223 154 0265 154 0266 Gramicidin_s Gram C60 H92 N12 010 1141 4697 1140 7054 1140 7059 Human_ins C257 H383 N65 077 S6 5807 6611 5806 6395 5803 6377 Hydroxyphenylazobenzoic Haba C13 H10 N2 03 242 2346 242 0690 242 0691 Hydroxypicolinic_acid Hpa C6 HS N 03 139 1107 139 0269 139 0269 InsulinB_chain Ins_b C157 H232 N40 047 S2 3495 9482 3495 6496 3493 6435 snenbenhalin Ten r28 H37 NS N7 Soc 4229 sos JAAN SoS 2693 off gt Figure 38 2 Compounds window There are currently eight categories of compound supported by the Compounds database These are General compounds any type of compounds non specific Amino acids Sugars Protecting groups for amino acid sequences N termini C termini Cations Nucleotides Definition of a general compound If a compound does not belong to a specific group i e nota sugar or amino acid then a general definition can be created by the following method Introduction 571 572 Chapter 38 Creating a compound database Introduction Set the Category to General compound and click on the New button The Edit Compound window will be displayed with the General compound property page shown as in Figure 38 3 General Compound Amino Acid Sugar Protecting Group Terminal Group Cation Nucleotide Name Formula E Cancel Figure 38 3 Edit Compound window for General Compounds Type in a name f
192. a Mass av Mass m a Mass mono Deoxyhexose Deoxyhex C6 H10 04 146 1432 146 0578 146 0579 Deoxypentose Deoxypent cS H8 03 116 1169 116 0473 116 0473 Heptose Hep C H12 06 192 1688 192 0633 192 0634 Hexosamine Hexn C6 H11 N 04 161 1579 161 0687 161 0688 Hexose Hex C6 H10 05 162 1425 162 0527 162 0528 Hexuronic_acid Hexa C6 H8 06 176 1259 176 0320 176 0321 Kdo C8 H12 07 220 1791 220 0582 220 0583 Muramic Mur C11 H17 N 07 275 2589 275 1004 275 1005 N_acetylhexosanine Hexnac C8 H13 N OS 203 1952 203 0793 203 0794 N_glycolylneuraminicacid Neu C11 H17 N 09 307 2575 307 0902 307 0903 Pentose Pent C5 H8 04 132 1162 132 0422 132 0423 Sialic_acids Sa C11 H1 N 08 291 2582 291 0953 291 0954 4 Figure 38 10 Example of an alphabetically sorted category To edit an entry in the list simply double click the mouse SELECT button on the required entry the Edit Compound window for that category of compound will be displayed To delete an entry from the database select the entry so that it is highlighted and press Delete Introduction 581 Chapter 38 Creating a compound database 582 Introduction Chapter 39 Searching for molecular weight matches Chapter 39 Searching for molecular weight matches 583 584 Chapter 39 Searching for molecular weight matches Pm f Programs gt Documentation Archiver se Favorites fa a z 5 5 3 Compounds Database L T Accessories A Documents pau 2 Configur
193. ak labelling 407 408 Chapter 22 Manual peak labelling Introduction Introduction There may be instances where the peak mass assignments carried out automatically by the MALDI MS program appears to be inconsistent with an expected mass value This may be due to impurities in the sample the method of sample preparation etc which gives rise to a distortion of the peak shape This will then lead to the peak centroid being weighted by artefacts caused by these factors In other circumstances it may be the case that the peak is simply too small to be identified below a significant threshold and automatic centroiding does not assign a mass to the peak In both of the above cases peaks can be manually assigned by using the Manual Peak Assignment window MALDI MS provides you with two methods to manually assign peaks e Peak labelling Manual peak assignment Chapter 22 Manual peak labelling Peak labelling This feature provides you with the a toolbar that allows you to add and remove labels from a spectrum You can also delete a range of masses for example matrix peaks Displaying the toolbar 1 On the spectrum right mouse click a menu list is displayed 2 Select the Peak labelling option another menu list is displayed Sint 931 mv sum 54900 mw Profiles 1 59 Smooth Gauss 10 Baseline 30 Insert Delete gt Annotate Wes Atiinobalion Properties i 102 9 i i Peak
194. alibration at a later date enter the name of the calibration required in the Name entry before pressing the Fire button to collect new data A list of available calibrations calibrations which have been saved is available by pressing the List button This list Figure 27 15 shows calibrations suitable for the currently displayed data or for the next data which will be collected For example if data is currently displayed for the data set selected on the Calibration window and it was collected in linear positive high mass mode but the instrument has been put in Reflectron mode setting List for to Selected data set will give a list of calibrations which were created from linear positive high mass Calibration of the instrument Chapter 27 Instrument Calibration Setting List for to Next data to be collected will list calibrations which were created from reflectron positive high mass data because the next data collected will be in the current instrument mode settings from the Experimental technique window H Calibration Files 4X List for Next data to be collected Figure 27 15 Calibration files list If you are about to collect new data set List for to Next data to be collected and choose a suitable calibration file This calibration will be loaded automatically each time the Fire button is pressed do not press Load as this will modify the currently displayed data If you are displaying data for
195. ally concerned here with relatively large chemical species the calculations can be complex and time consuming as well as demanding on computing resources The calculations are initiated from the Spectra tab of the Peptide Settings window see Figure 42 29 below amp Peptide Settings Keyboard Display Fragmentation Match Peaks Nested PSD Spectra Simulated Spectra Parameters Simulated resolution 2000 a Auto mass range V Display Parameters Sum Fragments V Clear OK Cancel Chapter 42 Sequence Calculator Figure 42 29 Spectra tab of the Peptide Settings window Many of the parameters are similar to those on the Display contents window for a Distribution display At Simulated resolution enter a value which would be applicable under experimental conditions Tick the Auto mass check box if you want to let the algorithm calculate and restrict the mass range rather than use the value specified on the main MALDI MS window You can choose to Sum peaks into single Gaussian envelopes or plot all individual peak patterns superimposed All other parameters are as already defined above i e a peptide sequence an enzyme if cleavage is required and the ions which are to be monitored Roepstorff nomenclature should be used Press Generate Spectra to initiate the calculations progress is displayed in the bar at the bottom of the window As described above it is possible to attempt very intensive calculations
196. an profile generated to be of width channels The coefficients in the gaussian convolution are chosen so that their sum is 1 e Savitsky Golay takes the convolution of the data points centred around the current data point with a quadratic profile of width channels The software provides an easy way of setting the smoothing width to approximately the correct value Simply place a pair of cursors around one of the data peaks in the mass range of interest and import these into the peak cleanup window using the button within the smoothing parameters containing the cursors icon Smoothing collected data 241 Chapter 16 Cleaning up data The shape of the smoothing filters applied to the data is shown in Figure 16 5 Average Gaussian Savitsky Golay Figure 16 5 Shape of the smoothing filters 242 Smoothing collected data Chapter 16 Cleaning up data Subtracting the baseline There are two methods available to subtract the baseline from the spectrum Baseline subtraction e Adaptive threshold peak detection Baseline subtraction Baseline subtraction is the process of determining the size and shape of any raised baseline or signal background that may be in the data This is indicated by the fact that the data peaks are not resolved to the baseline and appear to float on some invisible signal above zero It is usually caused by the use of high laser power or a large amount of chemical noise The parameters and their effect
197. ancel 6 Select the required folder and type in a suitable file name The export will produce three files e img this the main file that BioMap uses e hdr BioMap support file e t2m BioMap support file 7 Click the Save button 536 Biomap Chapter 34 Batch processor XML export Chapter 34 Batch processor XML export 537 538 Chapter 34 Batch processor XML export Introduction Introduction If you use the Axima for automated analysis typically LC MALDI imaging you will create large amounts of data You can export this data for use in other applications for example you can export imaging data to BioMap for analysis The XML export processor allows you to export mass spectrometric data as either mzXML which is an XML eXtensible Markup Language based common file format for proteomics mass spectrometric data or e mzData similar to mzXML developed by The Human Proteome Organisation HUPO Both data formats provide a standard and widely supported way of transferring mass spectrometric data between proprietary systems You can use the either the XML export processor or the Windows command prompt to process and export the required data This section describes both methods Chapter 34 Batch processor XML export Using the Batch processor Accessing and setting the Batch Processor 1 Select Batch Processor from the MALDI MS programs menu on the Taskbar Figure 34
198. and release will move the stage five steps or 25 steps if the Shift key is being held down prior to the button being pressed A continuous move will occur while the button is held down the move will be at the slow speed or a fast speed if the Shift key is being held down prior to the button being depressed Stop stage movement Stops the stage moving Initialise the sample stage This is the same operation as described in step 1 Initialise stage Open camera window Activates the stage camera window so the user can see where the sample plate really is Get current stage position in steps Updates the status pane in the bottom right hand corner of the Align Sample Plate window The units are stage motor steps Chapter 13 Preparation for data collection Alignment References The following buttons only work when a reference has been selected from the list of references Table 13 2 Alignment References functions Icon Action ucts Move to reference Move the stage to the currently selected reference position If the plate needs aligning this operation may not result in the plate being in the exact position for the current reference Jf Set reference Use the current location of the stage as the position for the currently selected reference point The values in the Stage X and Stage Y columns will be updated after this operation At this stage the plate should be correctly aligned Alignment is confirmed by depre
199. aning up data 235 236 Chapter 16 Cleaning up data Introduction The quality of the data obtained from the instrument depends on a number of factors namely the preparation of the sample and its accurate positioning on the sample slide the calibration of the instrument so that mass measurement will be accurate and processing the data to reduce baseline noise and to improve the signal to noise ratio To clean up collected data we need to use the Peak Cleanup window Select Peak Processing from the Processing menu Figure 16 1 angio2_msms0006 p14r_msms0004 MALDI MS 0 Eg File Edit view Instrument Automation Processing Help oS j AES A al x Calibration Annotation Profiles fi 2 Masses f1 2 1040 1 gt i sing Chromatography Polymers Sequence Calculator Reference Editor Select Peaks Manual Peak Assignment Internet Search Figure 16 1 Processing menu The Peak clean up window is a tabbed dialogue with three tabs The parameters on the main Identification tab have 4 basic categories as follows see Figure 16 2 on page 237 Smoothing of the data to remove reduce the effects of high frequency noise Baseline subtraction to remove reduce very low frequency noise from the spectrum Peak detection e Peak reporting Introduction Chapter 16 Cleaning up data These basic categories will in many cases be all that are req
200. ant threshold method The second parameter is the baseline multiplier This parameter represents how much larger the threshold is than the baseline If one were to set the offset to 0 0mV and the multiplier to 1 0x then the threshold would track the baseline of the signal Increasing the offset and multiplier values changes the shape of the adaptive threshold curve The values can be set so that the noise falls just underneath the curve allowing the algorithm to identify peaks in the signal The parameter values can be set by hand by typing the required value into the relevant box Equally the values can be set by eye by using the cursor and pressing the Subtracting the baseline 245 246 Chapter 16 Cleaning up data cursor select button next to the relevant box For example if you require the multiplier to be changed such that the adaptive threshold curve passes through one particular point on the spectrum that point can be selected by pressing the middle button on the mouse Pressing the multiplier cursor select button alters the value of the multiplier by the correct amount The result of this action can be seen by pressing the Apply to button There is a connection between the adaptive threshold and the signal baseline The filter width used to generate the adaptive threshold is the same as that used to generate the baseline curve In general the value of the filter width should be larger than the peak width otherwise the thre
201. arent Report Experiment File Based v Save Method Load Method Figure 15 29 Method Editor Report Select whether to report a Mass List or a Sequence Report by including the relevant entry in the Report box Report filenames are governed by the selection made in the Report Filename box Reports are saved to either the data filename run used for the current method or the experiment filename ker of the current Autorun Method Editor 199 Chapter 15 Automated operation lon finder The Method Editor lon finder window allows you to define a list of mass peaks of interest and use the lon Finder feature to extract the corresponding intensities from a spectrum The feature allows you to import or generate a list of masses tolerances export the data for use in third party applications To access the Report window select the Report label from the tree as shown in below Method Editor default mtd 1 x Sample Method Sample Method default mtd Parent Ion Finder Vii Parent MY Acquisition Ton search template v 333 Raster e ess e Add entry iol Laser Firing Paste data Import data v Processing VIL Calibration Remove entries Remove all EK Peak Cleanup v DA Monoisotopic Peak Picker j MILES Peak Filtering Results file destination RR Mascot Folder Folder file J Applications C a Compare Sequence Filename I Auto select filename Oligo Analy
202. arged File format PC 7 Columns 1 a 119 Delimiter comma x Decimal places 0 a 145 Export i Profile Average Processed Peaks 2 11 248 Format Intensity x 2 8 4 Repott intensities as m T 317 371 368 355 333 305 274 241 209 J oe0006 Processed data Charged 26 Figure 33 4 Example of a single column export file 526 Exporting ASCII data Chapter 33 Exporting data and data displays Multiple column output placed the selected number of column entries on the sample line e g two data sets delimited mass range 10 column output Figure 33 5 default Processed data Charged eer O E oo Delimiter comma Decimalpleces 0 368 355 333 305 274 241 209 Export _ Headings Profle Joe0006 Processed data Charged Average Processed Peaks 26 26 25 25 25 25 26 28 29 31 ireny E 32 33 34 34 33 32 31 Report intensities as my I Save as Cancel Figure 33 5 Example of a 10 column export file The items of data which can be exported are Headings Profiles Averaged Processed and Peaks The Headings are the graph headings and Profiles Averaged Processed and Peaks the values contained within the sample buffer arrays bins for each trace type in the displayed mass range An example is given in Figure 33 6 of all available export data written to the exported ASCII file Exporting ASCII data 527 Chapter 33 Exporting data and data displays a a i a fo a default Profil
203. arious options and sliders on the MALDI MS windows Mass ranges number of profiles sample number of profiles to average and the like are all examples of parameters All of the window parameters and window positions can be saved or loaded in one operation This means that operators can load their preferred environment at the start of a work session and save their instrument setup afterwards The dataset name is the general name under which all collected data is stored Each individual acquisition of data from a single sample is called a run Each run is automatically allocated a unique run number when data collection begins This run number is an incremental number appended to the chosen data name e g the first set of data acquired with the data name Joe would be J oe0001 Subsequent runs with the same dataset name will increment the run number e g the sixteenth run will be stored with the data name Joe0016 About data on MALDI MS Chapter 8 Loading and unloading data Loading data To load data into the MALD MS program either select Open from the File menu or click on the load data button on the toolbar angio2_msms0006 MALDI MS File Edit Yiew Instrument Automation Processing Help Display Spectrum hd Saver Girts Save As Open Auto Experiment Results Comments Parameters Print Ctrl P Prine Report 4 Print Preview Print Setup Export The
204. arrow between two range cursors containing the mass difference between the cursors with lines dropping to the graph baseline Annotate with an arrow between two range cursors containing the resolution between the cursors with lines dropping to the graph baseline Annotate with an arrow between two range cursors containing text with vertical lines to the full graph height Annotate with an arrow between two range cursors containing the mass difference between the cursors with vertical lines to the full graph height Annotate with an arrow between two range cursors containing the resolution between the cursors with vertical lines to the full graph height Chapter 20 Managing Data Displays Annotation with a line To annotate with a line place the cross hairs of both graph cursors at the start and end points of the line to be drawn and press Figure 20 37 phata SNA 100 110 120 Mass Charge J 100 110 Mass Charge Figure 20 37 Adding a line to a graph Annotation 385 Chapter 20 Managing Data Displays Annotation with arrows Horizontal arrows can be drawn on the graphs to mark mass differences fragment losses and the like Arrow lines are drawn horizontally at the position of the higher of the two cursor crosshairs To annotate with arrows place the cross hairs of both graph cursors at the start and end points of the arrow line and press Figure 20 38 100 110 Ma
205. ass list txt My Recent Documents Desktop 2 My Documents My Computer n ETE File name 7 peptide mid z Places Save as type Text Files txt z Cancel Figure 19 13 Saving a settings file 2 Navigate to the required folder and type in the filename for your results file 3 Select the Save button Saving and loading lon Finder settings 329 Chapter 19 lon finder Load your settings 1 Select the Load new button 2 Navigate to the required folder and highlight the required filename 3 Select the Open button settings are loaded 330 Saving and loading lon Finder settings Chapter 19 lon finder Processing the data This process uses the data within the Mass Tolerance fields and searches the currently loaded spectrum for the target ions For each ion peak the intensity is recorded The results are produced in a text file You can set the delimiter tab or comma of the results file within the lon Finder settings window see Importing data on page 321 1 In the MALDI MS window ensure that the required spectrum is displayed 2 Select the Process button a text file is produced with the results Interpreting the results An example of a results file follows P 7 peptide mix date070207_154103 txt Notepad File Edit Format View Help eee C Documents and settings Administrator Desktop mass ists peptide mixCdated70207_154103 txt Source file c Progra
206. at could result in serious injury or death Cautions highlight situations that could result in personal injury or damage to the Axima Where applicable specific cautions are included in the subsequent sections WARNING JfHigh voltages Do not remove any panels from the Axima the instrument can produce lethal voltages Do not modify the Axima Keep liquids and flammable vapours away from the Axima WARNING Electric shock The power supply must be suitable for that on the rating plate on the rear mains panel The power supply must be earthed Do not stretch twist or coil the power cable Do not remove any panels from the Axima the instrument contains a Class 3B laser Do not restrict or block the airflow at the back of the Axima the instrument may over heat resulting in a fire The Axima Resonance and QIT models have additional fan inlets outlets on the left and right hand sides Do not restrict or block the airflow Health and safety precautions Chapter 2 Safety warnings There are no serviceable parts Do attempt servicing Only Shimadzu or Kratos trained service engineers are allowed to service the Axima Precautions while using the Axima User instructions If the Axima is not used for the purpose which it was intended for any protection will be impaired Axima PC The Axima PC is for the control of Axima data processing only Do not install any other software without consultation
207. ate is selected Warning 1010 Range cursors not set on chromatogram Place range cursors on the Chromatogram before attempting to use a feature which requires them Warning 4000 There are no references to be saved Enter a set of reference points Warning 4010 There are no reference peaks to use in the calibration Check the calibration there appear to be no valid reference peaks in the calibration file Warning 5000 If comments are written into this directory the archiver will not be able to see them To ensure comments can be archived save relative to folder Comments can be stored here but it is recommended to put the comments in a subfolder of folder The Archiver will only look in the Comments folder registered with the Configuration Editor Warning 5010 Template cannot contain wildcard characters The wildcards and are not permitted Warning 6000 The requested parameter set could not be opened The defaults have been loaded instead Check that the requested parameter set file exists in the Parameters directory Failure to open the file has resulted in a set of default parameters being used The default values will allow the program to operate normally Warning messages Chapter 44 Summary of error messages Warning 6010 If parameters are written into this directory the archiver will not be able to see them To ensure parameters can be archived save relative to folder Parameters can be stored here but it is recommende
208. ation peaks Experimental approach For a given sample the experiment is as follows 1 Detect the primer and modification peaks within a specified mass tolerance 2 Measure all the peak areas 3 For each modification peak calculate the percentage area of the primer peak area 4 Calculate the summed modification peaks area as a percentage of the primer peak area 5 Estimate the signal to noise ratios for the primer peak detected 6 Calculate the primer peak resolution The sample is acceptable PASSES if the results of test 3 above are less than a specified set of values and if test 4 is less than a single specified value otherwise the sample is unacceptable FAILS Additionally the sample primer peak signal to noise ratio as measured in 5 and resolution test 6 must attain an acceptable specified value to PASS Lower limits are also specified below which the primer FAILS Between these limits the result is UNCERTAIN and the peak is classified as having poor signal to noise or poor resolution Method Editor 187 18 Chapter 15 Automated operation Experimental setup The greater degree of automated action introduced with the Axima series of instruments has already been described The Oligo Analysis experiment has been incorporated into this automated mechanism it is assumed here that the user is already familiar with the running of automated Axima experiments Select a suitable acquisition method i e inc
209. ave the same base name as the first larger parent mass file in the subtraction The software will generate extensions to this name as follows 1 similarities peaks found in both spectra 2 pos_differences peaks found in the first file but not the second 3 neg_differences peaks found in the second file but not the first The amino acid losses are displayed in the box to the right of the Subtract files button To attempt sequencing first identify a suitable start mass peak by placing a cursor close to it on the spectrum display ensuring that the correct trace is selected for processing in the Display contents window In the lower portion of the Nested PSD tab Select either N or C terminus and one of the direction arrows to indicate the search direction from the start mass Enter a suitable tolerance for amino acid mass matching and tolerance unit then Press the Generate button Amino acids identified appear in the list box in order of highest average score across the chain the number after the amino acid short code indicates the number of supporting evidence peaks found for the amino acid When sequencing the software makes assumptions about the type of fragment ion it finds and then looks for other related fragments which it regards as supporting peaks For N terminus sequencing these assumptions and supporting peaks are A 17 A B 17 B and C ion types and for C terminus sequencing they are X Y and Z The spectrum may
210. base WINdOW ccccceee eee e eee teense eee ee nee eaeeeeaes 36 File MON es seewdcs exe ceceeas irine n aa aE eras eebeeced eal EE i 37 VIEW MQNU 2 eect eee e etna 40 INStrOMeNnt MENU xine vatican deli viedo adele EEANN 42 Automation MENU 0 cccce eect eee ete eee ene e ene neta teeta 46 PrOCESSING Mensies nanie a a aS a 48 Help MENU csini i a 54 Graphical display SUD WiINdOWS ssssssssssssssrrsssrirrsrerrressrrrrerns 55 Display contents WINdOWS ssssssssssssssrrserrrresrerireserrrrererrrnee 56 Configuring Launchpad cceeseeeeeeeeeeeeeeeeeeeeeeeeeeeeueeeaueeeaeeaneeeans 59 Environment Configuration Editor ssssesseesssesereerrerrerrrrrnes 60 Mascot SeEtUp ira aaaea N a eA AEA E AEA A ues 62 The Log WiINdOW ccccceeeeeeeeeeeeeeeeeeeeeeuaeeeeuaeeeuaeeeeaeeeuaeeeeaeeauenennene 67 Windows event Viewer sssssssssssrrrsserrrssrrrresrerrreesrrinrnsrre re 68 Events logged sersniirnnm innon a aa AE 70 Loading and unloading data sssssssssssunnnnunnnnnnnnnnnnnnnnnnnnnnnnnnn 73 About data on MALDI MS cece cece ee cette eee eee eee eaten 74 LOADING data sieve ered s cede eee ee eieieid de neta erent dete ereras oe 75 UNO AGING datassa eed eis eaten ed Selene hae eee ee one dre eae eal 78 Parameter Sets sssssssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 79 TMLFOGUCHON orinn aa Vasdaaeadonatnennadbaaaadecaeaaaananines 80 Opening and saving parameter SetsS
211. base for possible matches open the Load Sequence window select the database to search and from the list select the database entries to compared Apply these selections to the Method by selecting the OK or Apply button When performing a search the select peaks parameters must be set for the current Method see Peak Cleanup on page 176 When selecting peaks avoid peaks at low mass e g below mass 300 as low mass fragments often occur at the same masses in digestions of peptides In other words the presence of such fragments is seldom of any use in identification of a particular peptide Chapter 15 Automated operation The results of the search are saved to a file for reviewing This file is named after the current Method and stored to the sequence report directory beneath the default Method directory e g C Program Files Shimadzu Biotech Launchpad method sequence report default txt The report can be automatically printed see Print on page 194 Automated Quality Analysis of Oligomers The degradation of oligomer samples follows predictable pathways The purity of an oligomer sample is inversely proportional to the concentration of degradation products The Axima instrument can be used to detect the characteristic peak mass of the primer peak and the peaks corresponding to a number of mass losses or gains from the primer mass the purity can then be estimated in terms of the relative intensities areas of these modific
212. be displayed with the Amino Acid property page shown as in Figure 38 5 General Compound Amino Acid Sugar Protecting Group Terminal Group Cation Nucleotide Name PO BB Index f0 Lon Short Class Aliphatic x HPLC Index 0 Eoma 0 wr CW Inde fo pwz ESGIndex O Average Mass Colour a KD Cl Most Abundant Mass Apply Cancel Figure 38 5 Edit Compound window for Amino acids Introduction 575 576 Chapter 38 Creating a compound database Introduction This window displays parameters required to define an amino acid It shows the amino acid name long and short symbols along with the elemental formula of the amino acid residue The entries are as described below Name This field should contain the most commonly used name for the amino acid Long This field should contain the long abbreviation generally used for the amino acid These generally contain three letters but may contain more Short The short symbol field should contain a single letter abbreviation for the amino acid All the short symbol fields in the database must be different Lower case letters will be treated as different symbols to upper case letters Class Seven different classes are allowed Aliphatic Side Chain Aromatic Cyclic Basic Acidic and User Defined If this field contains any other string the class is coded as Unknown Formula The elemental formula field contains the elemental formula for the amino acid All elem
213. bill KRATOS My Documents iTRAQ Results iTRAQ 2 1 117 114 80 00 08 03 1047 0001 run Pi C Documents and Settings bill KRATOS My Documents iTRAQ Results iTRAQ 2 1 117 114 80 00 08 03 1047 Im20001 run E Ly VOU Dannumanke and Caktinacthill VN ATACA RactinmamkliTA AON medkhiTO AO 2 4 147 114409 NN 90 94 1290 NAM vim Remove Clear Destination Folder Same as source Specific folder Bronse Process Files Z 542 Using the Batch processor Chapter 34 Batch processor XML export Figure 34 5 Batch Processing window with run files 5 You can add further files remove files highlight the file and select the Remove button or clear all the files from the list select the Clear button Processing the files 1 Select the destination folder for the processed file e Same as source will place the generated processed file in to the same directory folder as the source files Specified folder select the folder where you require the generated processed file 2 Select the Process Files button a 22 Batch Processor ioj x oe Generating c documents and settings bill kratos my documents itraq results 6 protein mix 2007030 File Progress E C Documents and Settings bill KRATOS My Documents iTRAQIR Completed E C Documents and Settings bill KRATOS My Documents iTRAQIR Completed E C Documents and Settings bill KRATOS My Document
214. by using cursors Chapter 16 Cleaning up data Peak Reporting Peaks are either reported as their Centroid Gradient Centroid and Threshold Centroid and Gradient 25 Centroid and Threshold 25 Centroid or their Apex Threshold Apex In the case of the centroid calculation the software calculates the weighted mean mass based on the signal intensities between the peak limits In other words the peak centre is defined as the mass at which the area between the start of the peak and the peak centre equals the area between the peak centre and the end of the peak In the case of apex peak reporting the mass of the peak is simply the mass associated with the largest amplitude time bin within the peak limits In general the centroid method will yield the most accurate peak centres and will correspond more closely with average mass calculations where the isotopes of a distribution have not been resolved While the apex value may give better results if the peak signals are affected by noise or in the event that unrelated peaks have not been resolved correctly Peak reporting is shown in Table 16 1 on page 252 only those with width greater than the peak width parameter are accepted Note that in the case of Gradient 25 Centroid and Threshold 25 Centroid the 25 isa threshold value i e only the top 75 of the peak is used during the centroiding process Peak Reporting 251 Chapter 16 Cleaning up data Table 16 1 Met
215. c residues can be highlighted in a user defined colour Seven categories can be specified using the colour map 1 Colour Each amino acid is allocated a different colour code defined in the amino acid database Chapter 42 Sequence Calculator 2 Class The amino acids are divided into eight classes each of which is given a different colour code Default settings are detailed below Table 42 1 Colour codes for different amino acid classes Class Colour Aliphatic Cyan Side chain with hydroxyl or sulphur Yellow group Acidic Blue Aromatic Green Basic Red Cyclic Magenta User Defined Grey Unknown Black 3 BB The Bull Breese index for the peptide is colour coded Reference 2 This index is a measure of the partition between an aqueous and hydrophobic phase These values can be related to surface matrix activity during sample ionisation In general the more hydrophobic the peptide the more dominant the spectrum becomes in the presence of a less hydrophobic species Peptides with a negative index correspond to hydrophobic behaviour Hydrophilic peptides have a positive index A thorough discussion of the subject is given in Reference 2 A number of alternative measures of hydrophobicity are available in place of the Bull Breese index These are 4 HW Hopp and Woods Reference 4 KD Kyte and Doolittle Reference 7 6 ESG Engelman Steitz and Goldman Reference 3 A discussion of the subtle differences between these m
216. c cece ee eee eee eee eee teeta tena ee ed 398 Amplification 22 csc c cece etnecens cedeeee cient onde ence cena nd needa eedeeie ee et 401 Compound Database Viewer cccccceeeeeeeeeeeeeeeaeeeseeeneneneuenenenens 403 Manual peak labelling ccscsscssscsceeceseeseeeneeeeeneeeeeneeeeeeneeeeeasease 407 PAMEMOGUCEIONY ffens5 da i eieaeua vated neo dea TE 408 Peak labellinG csscccis set catenseed eteaees ids de ead ceender Ea a E 409 Manual peak aSSiIQNMeN cece eee eect teeta teeta eee ened 412 Displaying Simulated data ccceeceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeenenens 415 PMEPOCUCTION cies dindecec cece eee ee eaten ss akeni niaaa ENA EENE 416 Displaying isotopic distributions cece eee eect ee teeta tees 417 Theoretical spectra for peptides cece eeeeee eee ee eee eee ened 420 Displaying reference files cece cece eee eee eee e teeta eee 423 Choosing user defined colour SCHEMES ss sssssssssnnnnnnnnnnnnnnnnnnnn 429 PMEROGUCEIONY 1352 cece deslamcncesaiteean ohedheaaa T pecameand aa bes 430 Changing spectrum trace COIOUSS cccceeeeeeeeeeeeeeeeeeeeeuaeeas 431 Changing chromatogram trace COIOUIS cece cece eee e eee 434 Changing distribution trace COIOULS eeeeeeeeeeeeee ee ee eaten ees 436 Changing COMMON COIOUSS cece cece eee eee eee teeta reren rnes 437 Changing cursor COIOUIS cceee cece eee eee ee teeta e teeta reren rnes 438 Chan
217. cations Power g r gt V amp S Compare Sequence i Oligo Analysis N h EW Output Profiles fio a per sample Wc Print 3 Export Shots or v accumulated per profile A Report VEI MS MS eee Laser Rep Rate 10 0 ME Acquisition Peak Selection gt Mass Range Mode v RH Raster Low Low Mid High Hi v3 Laser Firing 1005 300 8509 2k 3k El MY Processing l EK Peak Cleanup ZIM Monoisotopic Peak Picker VIA Peak Filtering vS Mascot Yi Output X gt Data Storage Get Manual Settings Save Method Load Method Figure 15 9 Method Editor QIT Laser Firing in MS mode Laser Firing I Restrain peak cleanup For Axima Resonance instruments select the mass range of interest by selecting from one of the 5 mass range buttons These correspond to the 5 modes of instrument operation as set up in the factory Method Editor 173 174 Chapter 15 Automated operation Method Editor default mtd Sample Method Sample Method default mtd iy MS VY Acquisition v 333 Raster VX Auto Quality 3 Laser Firing QIT v Processing VIL Calibration Peak Cleanup VUE Monoisotopic Peak Picker VIA Peak Filtering VS Mascot v Applications vA Compare Sequence M Oligo Analysis v Output VQ Print Vi Export vU Report lon Finder vI MS MS Vy Acquisition BE Peak Selection M fa Raster 3 Laser Firing Processing EK Peak Cleanup VI i Monoisotopic Peak Picker VEA Peak Filtering VS
218. ccept the suggested defaults during Launchpad software installation the Home directory will be located at C Program Files Shimadzu Biotech Launchpad and the other folders such as data calibration etc will be located there as sub folders Some users find it convenient to store MALDI MS files at several locations across a network and the Archiver can be used to locate select and archive data in such cases E Archiver x Restore from JEstore Help Compress Decompress Refresh Clear selections C NetMeeting J Online Services C Outlook Express J Program Shortcuts J Quadralay E E RoboHelp Office J Shimadzu Biotech Launchpad E J Calibration ey Data C 04 March 05 J Andy JI Bil C Default O linear mess E Mascot C Robert Lane C Databases J Experiments J Internet 4 Log ha File information File totals a 4 E Show file information IV Figure 32 2 Archiver window Please note Any files to be compressed or decompressed must not be currently loaded in the MALDI MS program Before processing these files with the archiver unload them in the Load window see Unloading data on page 78 Introduction 517 518 Chapter 32 Archiving data Introduction Individual files within folders or complete folders can be selected by ticking the box next to the icon for the item Items ticked will be selected for archiving Figure 32 3 To deselect an item simply clic
219. ce Ctrl F Links Ctrl L Insert panel Delete panel Clear markers Delete sequence Figure 42 12 Popup Edit menu When starting a new sequence in an empty panel be sure to click the mouse SELECT button in the empty viewing area to obtain an insertion point s before inserting an amino acid from either the Introduction 613 614 Chapter 42 Sequence Calculator Introduction Sequence Keyboard window or the computer keyboard Note that the amino acid short symbols are case sensitive so that for example the letters a and A may stand for two different amino acids Amino acids are inserted or deleted from the current insertion point which is the position marked by a small triangular symbol s To change the insertion point click SELECT in the viewing panel at the new insertion point The popup edit menu functions are described in Table 42 2 below Table 42 2 Popup edit menu functions commen Action d Cut Remove the current selection and place it on the clipboard Copy Copy the current selection to the clipboard Paste Paste the contents of the clipboard at the current insertion point Find and Find a given sequence within the selected replace viewing panel Protecting Attach a protecting group to the selected unit in groups the sequence Links Create a cross link at the selected unit in the sequence To select a delimited sequence of amino acids click SELECT on the first amino acid and click Shift SELECT o
220. collected in linear mode or Mass to Low to list only data collected in low accelerating voltage mode Using Any as a match setting displays data with any setting for that specific option P Ext specifies whether pulsed extraction was On or Off for the listed datasets Gate can be set On or Off or to Blank The list can be sorted in name or date order depending upon individual requirements Name and title matching can be used to list only files which match a specific name or title To list all files which contain the letters Ma as part of the name switch the match option to Match as opposed to Any then simply type Ma in to the Name entry which appears adjacent to the selection and the list will display all data which contain these two letters e g Martin Malcolm AlMa etc The list can either display the title line typed into the comments window or display the comment for the first sample spot from which data was collected Set Display to the required option Select a filename from the File browser window list and the selected filename will be copied to the currently selected slot on the Load data window Press the OK button and the selected data file will be loaded the loaded dataset names will be displayed on the top frame bar of the base window Figure 8 4 Data name Peal Frame bar ff angio2_msms0006 p14r_msms0004 MALDI MS Oy x File Edit view Instrument Automation Processing Help slal s eB ja 8
221. cross hairs Pata el SEAS 100 110 120 Mass Charge rf D g Pat sal al el 100 110 120 130 Mass Charge 388 Annotation Chapter 20 Managing Data Displays Figure 20 40 Adding a boxed region to a graph Annotation with cursors Range cursors can be used to annotate the region between the cursors The annotation can include a mass difference text or centre mass mass difference m Am Annotation using cursors takes the form of a horizontal line between the two cursors The horizontal line is always drawn at the higher of the two cursor cross hairs Vertical lines can be drawn at both ends of the horizontal line either to the full height of the graph or up to the horizontal line In both cases the vertical lines original from the bottom axis of the graph The mass difference Am between the two cursors can be shown alternatively a measure of the resolution m Am can be applied to the display The number of decimal places used in labelling numbers is set with the Decimals option on the New Annotation window On the display position the range cursors at two points on the graph between which cursor annotation is to be marked and select the required option from the New Annotation window Figure 20 41 Annotation 389 390 Chapter 20 Managing Data Displays jas K 10 85 100 11 Mass Charge K 12 18 11 Mass Charge Figure 20 41 Thre
222. cular weight 244 2942 The matches can be stepped through one at a time using the next previous match buttons or a match number can be typed into the Match entry Elements and compounds can be added to or removed from the search list Click the mouse SELECT button Chapter 39 Searching for molecular weight matches on an entry in the search list to edit that entry press Insert after typing in a new formula or quantity or Delete to remove the selected entry To clear the whole list press Clear list and the list will be emptied ready for a new list to be entered Chapter 39 Searching for molecular weight matches 588 Chapter 40 Polymer simulation Chapter 40 Polymer simulation 589 Chapter 40 Polymer simulation Introduction The MALDI MS software suite has a facility for simulating the spectra resulting from polymer series There are occasions when the polymer chemist is aware of the likely composition of a polymer and would like to simulate its mass spectrum By comparing the simulated mass spectrum with an actual mass spectrum confirmatory identification is a simple process The Polymers window allows polymer series to be generated and used as a reference file so that spectra for the material can be simulated To start the Polymers window select Polymers from the MALDI MS programs menu on the Taskbar Figure 40 1 Launchpad Click the Polymers icon Info Show on startup V _Heb gt B
223. d DE s OK Cancel Figure 42 21 Fragmentation tab of the Peptide Settings window Calculating sequence masses The Fragmentation tab of the Peptide Settings window controls the overall structure of the report It is possible to generate a report based on the whole peptide or a delimited range of amino acids within the peptide If a portion of the sequence is highlighted then a report will be generated only for the selected portion Otherwise it will be generated for the whole sequence The report will calculate masses on the basis of Most Abundant or Average or Monoisotopic distributions as selected by the Masses entry on the Sequence Calculator window A peptide sequence can be digested with a specific enzyme reagent to produce digest fragments which will be seen in a spectrum of the peptide By selecting Method on the Digest Introduction 621 622 Chapter 42 Sequence Calculator fragments panel the fragments can be theoretically predicted and their expected positions within the mass spectrum marked to aid in identification of the fragments in the spectrum Digest of peptide sequences The use of enzymes to cleave large peptides is a well established method for structural validation Highly specific enzymes are available which cleave the peptide chain according to simple rules The same applies to certain chemical reagents such as cyanogen bromide which degrade the peptide in a well understood manner The
224. d and the previously saved scheme will be loaded Press Load defaults to use the factory defined MALDI MS colour scheme Changing common colours 437 438 Chapter 24 Choosing user defined colour schemes Changing cursor colours There are up to 10 cursors available on the MALDI MS base window displays there are used to display multiply charged fragment positions dimer trimer positions etc To differentiate between the cursors their colours can be user defined The Colour Editor for cursors is shown on the Cursors property page of the Display Options window Figure 24 8 Display Options Ci x General Graphs Graph Text Text Report Cursors Peak Labels Type Normal 7 Tolerance tooo ppr F Mass upto E Width None Normal Display Window Height Graph Display window Join with Off Line ion Gate Colours T Al displays ok __Appy Cancer Figure 24 8 Colour Editor button on the Cursors property Changing cursor colours page Chapter 24 Choosing user defined colour schemes Select Colours from the Cursors property page the Colour Editor Cursor window will be displayed as in Figure 24 9 Colour Editor Cursors SBE Mass Tolerance Additional mass and charge cursors i 02 a eal ee BB i ee ee Colour sets Load Load defaults Apply Figure 24 9 Colour Editor for Cursors The Mass and Tolerance cursor colours define the movable cursors
225. d are filtered using the Category option When a cursor is placed on the spectrum the upper list highlights the closest entry in mass to the current cursor position Above the list is an indication of the mass difference between the database mass and the actual mass under the cursor A negative value for dM indicates that the cursor mass is lower than the database entry and vice versa When two cursors are present on the display the lower list highlights the closest entry in mass to the mass difference between the cursors In this case the upper list shows the closest entry in mass to the last moved cursor position Chapter 21 Compound Database Viewer The mass or mass difference can be annotated on the selected display by clicking the mouse MENU button in the list containing the highlighted entry A popup Annotate option will appear Clicking on this option will apply the highlighted database entry as a marker on the selected display Figure 21 2 Eile Edit Yiew Instrument Automation Processing Help kif 2 Ee Bl E x x Display Spectrum v Profiles 1 Masses 649 1395 l gt I Data p14r_msms0004 4 Mar 2005 13 47 Cal tof 4 Mar 2005 10 10 CID Shimadzu Biotech Kompact MALDI 6 V2 7 0 Beta 1 Mode reflectron_ms_ms Power 74 Gate 1520 00 1545 00 P Ext 1534 lnt 6 0 m sum 1325 mV Profiles 25 245 Smooth Av 1 1357 18 100 804 964 99 jogs gg 1163 18 1259 85 663 67 758 6 854 4 7a ans 24
226. d mouse configuration where windows has been configured for left handed operation of the mouse then the buttons should be reversed ADJ UST SELECT MENU Figure 3 2 The mouse buttons for right handed operation You should familiarise yourself with the Windows environment and the use of the mouse keyboard and windows by referring to the on line help and tutorials installed with the operating system The Launchpad suite of programs will have been loaded into a folder on the PC either pre loaded in the factory or loaded by the installer and we assume that the application programs have been loaded into the default folder C Programs Shimadzu Biotech Launchpad From this point we will refer to this folder as the Launchpad programs folder however the exact location of this folder may vary depending on where the user chose to install the software Each of these programs has an icon associated with it Table 3 1 on page 25 shows the icon for each program within the Launchpad suite and a brief description of the program function Looking in the Launchpad programs folder with the Windows Explorer will show the icons in Table 3 1 double clicking on the icon will launch the respective program Table 3 1 Description of the applications Program Function MALDI MS allows you to perform a MALDI MS experiment and control the Axima Search program allows you to search for elemental combinations which match a given mass fo a Introduc
227. d on an Axima instrument When entering formulae enter the standard atomic symbol for each element followed by a quantity for that element A space or full stop period can be typed between the element symbols to make the formula easier to read see Editing the Element Database on page 566 Theoretical spectra for peptides 2421 422 Chapter 23 Displaying simulated data E g H2 O for water Repeated formulae may be entered by using brackets E g C6 H2 O4 10 is the same as C60 H20 040 See the section on creating a formula database for further information on typing in molecular formulae The Gain or Loss option allows adducts to be added or subtracted from the molecule A loss of H is common when negative ions are produced Press Clear to clear all settings in the window Theoretical spectra for peptides Chapter 23 Displaying simulated data Displaying reference files Reference information can be shown either as a graph or a text listing of masses and formulae Reference files are used for calibration see Calibrant reference files on page 461 for details of creating reference files for calibration purposes Displaying spectra for reference compounds Reference files can be displayed as if they were data collected from the instrument This allows the user to match positions and shapes of the reference peaks with those obtained on slides containing samples with internal calibrants To create a reference
228. d the popup menu shown in appears The current setting will be indicated with a tick simply choose the required setting and release the mouse H Calibration r Calibrant references E pl41_msms0004 G7 List references Reference editor Compounds Mass Formula Abundance Cursor mass Time CSO H72 N13 012 96 69 C62 H90 Ni O14 it 1533 86 C76 H113 N18 016 I 1800 94 C87 H126 N21 021 I 2093 09 C95 H146 N29 023 2465 20 C112 H166 N27 036 I 3657 93 C167 H258 N47 046 I Average Mass Most Abundant v Monoisotopic Distribution Figure 27 12 Toggling the mass setting Calibration text reports After calibration the list of mass time values can be seen by selecting Calibrant list from the Display option on the base window Figure 27 13 This list shows the reference masses which were matched with sample peaks in the calibration and the times at which those peaks arrived at the detector Data p14r_msms0004 F 1 c 4 Mar 2005 13 47 Cal tof 4 Mar 2005 10 10 CID Shimadzu Biotech Kompact MALDI 6 Vv2 7 0 Build 20050509 Mode reflectron_ms_ms Power 74 Mass Time 379 0930 39367 0000 1533 8580 79140 0000 Figure 27 13 A Calibrant list report This report can be generated for any loaded data set by selecting the data set on the Display contents window Figure 27 14 fA Calibrant List Contents BBE Dataset Trace Sample fea 1 angio be e a Figure 27 14 Display contents window f
229. d to put the parameters in a subfolder of folder The Archiver will only look in the Parameters folder registered with the Configuration Editor Warning 7000 The Name template cannot contain wildcard characters Warning 7010 The Title template cannot contain wildcard characters Warning 7020 The Find template cannot contain wildcard characters The wildcards and are not permitted Warning 8000 This dataset is already loaded Warning 8030 Duplicate data sets have been selected removing dataset The requested dataset already exists within MALDI MS it therefore ignores the request to load the dataset a second time Warning 9000 The raw file for this data is empty Warning 9010 The stats file for this data is empty The files may have been corrupted or data collection may have been terminated before the files were written to Warning 10000 Could not stop internal timer used to drive the system The application may not continue to function correctly Warning 10010 Could not restart internal timer used to drive the system The application may not continue to function correctly Shutdown MALDI MS and restart if the problem continues shut down Windows and reboot There appear to be problems allocating timer resources used to control the instrument Warning 12000 Cannot allocate memory for copying a protecting group In all of the above cases close other programs shutdown active processes to free more memory
230. d using the Segments option This option serves the same purpose as the Segments option on the Display contents window for chromatograms For example setting Segments to 10 with Masses set to 1000 11000 would give ten regions with ranges 1000 1999 2000 2999 etc Peak detection would be carried out within each region and the results reported for all ten regions The Combine option permits overlapping profile regions with detected peaks in them to be combined into one region reducing the overall number of detected peaks Having selected the options required on the window press Detect peaks to start peak detection Viewing the detected peaks An example of data used for peak detection is shown in Figure 30 3 lnt 100 2 0 mV mass 3715 to 14680 10000 Mass Charge Figure 30 3 Example of chromatography data The settings used on the Chromatography window to detect peaks within this data from a continuous slide are shown in Figure 30 2 In the example peaks were searched for in the range 9 000 12 000 amu On searching this region four peaks were detected Chromatogram displays were used on the base window to view the detected peaks Chromatographic peak detection 507 508 Chapter 30 Chromatography The peaks found within the data will be displayed in the lower half of the Chromatography window Figure 30 4 Update Order Descending Ascending Profiles Mass Both Profile tagging Height Mass Pr
231. daries with the range cursors To use Manual peak assignment select a Spectrum display Processed trace and position two cursors on a peak so that they bracket the required limits of the peak Figure 22 3 On the Manual Peak Assignment window press the 4 button and the bracketed region between the two cursors will be centroided using the currently selected centroiding method on the Peak cleanup window The centroid mass will appear in the list of manually assigned peaks Manual peak assignment Chapter 22 Manual peak labelling 515 95 1005 515 95 315 95 T T T T T T s16 s18 520 522 512 514 516 518 520 522 1 Peak too small for automatic 2 Position cursors to bracket region peak detection to be centroided 15 95 15 95 o7 T T T T 512 514 516 518 520 522 3 Press button on Manual Peak assignment window and peak will be manually assigned Figure 22 3 Steps for manual peak assignment If the new mass is within 1 Dalton of any other mass assigned peak in the spectrum it will overwrite the existing entry this prohibits peaks appearing within 1 dalton of another mass assigned peak Manual peak assignments are retained regardless of any reprocessing carried out on the collected data The colour of the manually assigned peaks can be selected on the Display Options window Graphs property page Figure 20 29 on page 372 The only means of removing manually assigned peaks in by using the
232. data for the next and previous mass respectively The button would average the data for all masses and show this average 6 7 mV sum 955 mV Profiles 1 142 Smooth Av 20 Baseline 80 Wint 6 2 mV sum 875 mV Profiles 1 142 Smooth Av 20 Baseline 80 1534 33 21 34 Original view 1 Scroll to minimum mass ee Shift 1200 1400 1600 1800 2000 2200 2400 2600 200 400 600 800 1000 1200 1 MassiCharge MassiCharge Scrolling graphical displays 349 350 Chapter 20 Managing Data Displays wint 100 80 60 lnt 100 80 60 6 7 mV sum 955 mV Profiles 1 142 Smooth Av 20 Baseline 80 1534 33 Scroll left 10 E 1801 88 1000 1200 1400 1600 1800 MassiCharge 2000 2200 2400 6 7 mV sum 955 mV Profiles 1 142 Smooth Av 20 Baseline 80 Scroll right 10 1534 33 1801 88 1400 1600 1800 2000 MassiCharge 2200 2400 2600 Bint 100 Sint 100 80 60 0 0 mV sum 3 mV Profiles 1 142 Smooth Av 20 Baseline 80 6780 84 Scroll to maximum mass Shift 6670 45 6999 49 7400 7800 Ai A a 7200 Mass Charge 7600 8000 6 7 mV sum 955 mV Profiles 1 142 Smooth Av 20 Baseline 80 1534 33 Display full graph range 21 34 21 67 22 38 2000 4000 MassiCharge 6000 8000 Figure 20 10 Scrolling graphs using display toolbar buttons The range of the data displayed will be the same for each display For example Figur
233. detailed explanation of the Mascot Search Engine and its parameters refer to the supplied manual Mascot Installation and Setup or visit the website at http www matrixscience com Compare Sequence Method Editor The Method Editor Compare Sequence window provides functionality for peak matching the acquired data to a peptide sequence or a database of sequences see Sequence Calculator on page 601 To access the Compare Sequence window select the Compare Sequence label from the tree as shown in Figure 15 17 below Chapter 15 Automated operation Method Editor default mtd Sample Method MX Auto Quality Compare Sequence Me Laser Firing MEI Processing MILE Calibration ER Peak Cleanup FF Search Currently loaded sequence CTA Monoisotopic Peak Picker y q Select Peaks EN Peak Filtering Match Settings MA Mascot mM Appicaion Missed theoretical peaks IV Missed experimental peaks V Tolerance f2 a Da x r Compare Sequence Options M ag Oligo Analysis Sequence aa Guiput Font Medium 7 Colourmap Colour T Sequence Short symbol Me Print i O Export OW Report Settings C a lon Finder ME MS MS amp E VI Acquisition P Peak Selection YGGFLRRI B M Ea Raster 5 3 Laser Firing Vy Processing EE Peak Cleanup Ml Monoisotopic Peak Picker l 4 gt gt gt Name Untitled a MILES Peak Filtering 1 MAR Mascot N terminus Hydrogen H m WE Ou
234. digest products Introduction Chapter 42 Sequence Calculator These cleavage methods and their associated rules are stored in the enzyme database This is editable by the user with the Enzyme Database editor described in section on page 597 and hence customised digests with alternative enzymes can be accommodated by the software The Report option specifies the information to be included in the enzyme report The available items of information which can be displayed on the report are Fragment number Position Masses Bull Breese I ndices HPLC I ndices Elemental formulae and Sequence The Sort by option selects the sorting criteria for the enzyme digest fragments The fragments can be sorted by Sequence Position Mass Bull Breese Index or HPLC I ndex For instance selecting an HPLC index sort will arrange digest products in the order of elution from a reverse phase HPLC column Although enzymes cleave peptides at well defined positions it is possible to obtain fragments which arise from missed cleavages This is best explained by reference to a peptide chain diagrammatically shown in Figure 42 23 gt P gt b amp b Figure 42 23 Example of enzyme cleavage The peptide can theoretically be cleaved at four positions generating five smaller peptides A B C D and E This is the perfect mode of cleavage for a peptide However if we allow for a first level of missed cleavage we can generate the fragments AB BC CD
235. display set the Display option to Reference then click on the toolbar Display Contents button to show the Reference Contents window Figure 23 6 a Reference Contents BBE Data from EA List Traces Profile Peaks Auto mass range IV Resolution 2000 4 Apply Figure 23 6 Reference Contents window The Data from option specifies where the reference data is to come from If Data from is set to Calibration Window then the reference file selected on the calibration window see Instrument Calibration on page 459 will be used for the reference display The Reference and List options are unavailable when Calibration Window is selected Displaying reference files 423 424 Chapter 23 Displaying simulated data If Data from is set to Other Reference then the name of a reference file must be specified in the Reference entry The available reference files can be listed by pressing the List button When an elemental composition formula has been typed in for each reference peak peak profiles can be displayed The program uses the formula to create isotopic distribution profiles for the references Where a formula has not been provided only the Peaks display is available This will draw a vertical line at the mass position of each reference peak The Resolution option provides a means of simulating the profile displays at differing resolutions This allows simulation of data for dif
236. displays lon Finder This feature is described in a separate chapter see page 317 534 lon Finder Chapter 33 Exporting data and data displays Biomap Biomap is a software application used for analysing MALDI images and is available as a free download from the MALDI MSI web site at http www maldi msi org index php Exporting to a Biomap compatible file 1 Display the sample profiles you wish to export 2 Select File gt Export gt Biomap Ej Biomap Export m Mass Range Use the mass range from the spectrum display Use the mass range specified below Start mass Da 1000 End mass Da 3000 Intensity Scale intensities to largest Crop saturated intensities 3 Select the required mass range 4 Select the required intensity radio button e Scale intensities to largest the peak with the largest intensity is used to scale all the other peaks Crop saturated intensities peaks with intensities above 32 767 are cropped You may need to experiment to see which of these two radio buttons produces the best results within Biomap Biomap 535 Chapter 33 Exporting data and data displays 5 Click the Export button Save As BEG Save in E Data ce EE Fe My Recent Documents Desktop rA My Documents hos My Computer 1 enna File name l z Places Save as type fima t2m and hdr file img z C
237. ds Database window Introduction 570 Chapter 38 Creating a compound database This will display the Compounds window Figure 38 2 Category EGE feria Sort Alphabetic New Delete Export Help Compound Formula Mass fav Mass m a Mass mono ACTH_fragmenti_xvii ACTH_xvi C95 H145 N29 023 S 2093 4484 2093 0817 2092 0789 ACTH_fragmentxviii_xxxix ACT C112 H165 N27 036 2465 7087 2465 1937 2464 1911 Aldolase Ald C1733 H2773 N489 0525 S11 39211 8752 39210 9227 39187 2250 Angiotensin_i Angi C62 H89 N17 014 1296 4987 1295 6769 1295 6775 Angiotensin_ii Angii C50 H71 N13 012 1046 1972 1045 5340 1045 5345 Angiotensin_iii Angiii C46 H66 N12 09 931 1085 930 5071 930 5076 Anthranilic_acid Anth C H7 N 02 137 1384 137 0476 137 0477 Azothiothymine Att C6 HS N3 OS 167 1900 167 0153 167 0153 Bovine_insulin Ins C254 H377 N65 075 S6 5733 5615 5732 6062 5729 6009 Bovine_serum_albumin Bsa C2935 H4582 N780 0899 539 66430 0694 66427 4319 66386 5910 Bradykinin Brad CSO H73 N15 O11 1060 2273 1059 5609 1059 5614 Bradykinin_fragmenti_vii Bra C35 H52 N10 09 756 8620 756 3915 756 3919 Caffeic_acid Caff C9 H8 O4 180 1604 180 0422 180 0423 Carbonic_anhydrase Carb C1312 H1995 N359 0383 S3 29021 7330 29021 1728 29003 6830 Cyanohydroxcinnamic_acid Cya C10 H7 N 03 189 1708 189 0425 189 0426 Cytochrome_c Cytc C560 Fe H874 N148 0156 54 12360 1426 12359 7428 12352 3239 Dithranol Dit C14 H10 03 226 2322 226 0629 226 0630 Ferulic
238. e The Title appears at the top of each display for the dataset regardless of the sample spot number being displayed Introduction 85 86 Chapter 10 Putting comments with collected data The Prefix message is shown before each sample comment This message is also displayed for the dataset regardless of the sample spot number being displayed The Comment message appears adjacent to the Prefix message but only when the spectrum for that well is displayed Having typed in the comments they can either be applied to the Current dataset being displayed or to the Next data collected Adding comments 1 Open the Comments window File gt Comments 2 Enter the required fields use the table below for guidance Field Title Prefix Select well List Samples Comment Apply to Introduction Table 10 1 Comment fields Guidance What you enter here appears on the top line of the spectrum header What you enter here appears at the start of the second line Enter the well location The location of the current well appears after the file name in the header Data ang11__0001 021 Feb 2009 11 20 Cal tof 4 Mar 204 Shimadzu BA Mode reflectron Power 63 Blankeg Well location Allows you to define what well locations are displayed in the window Select from the drop down list Double click the required well What you enter here appears on the second line adjacent to what you entered for the Pre
239. e Name Formula Chapter 38 Creating a compound database Figure 38 8 Edit Compound window for Cations Type in a Name and Formula for the cation group then press OK or Apply Defining Nucleotides Select Nucleotide as the Category then click on the New button The Edit Compound window will be displayed with the Nucleotide property page shown as in Figure 38 9 below General Compound Amino Acid Sugar Protecting Group Terminal Group Cation Nucleotide Name Unit formula Unit name Backbone 5 end Cancel Figure 38 9 Edit Compound window for Nucleotides Re using previous definitions Complex formulae may be created by re using previously entered compounds as shown in Table 38 3 Table 38 3 Combined compounds Formula Compound name Asp Arg Val Tyr Ile His Pro Phe Angiotensin Arg Pro Pro Gly Phe Ser Pro Phe Arg Bradykinin AngiotensinI Bradykinin a A new compound can be created from any number of previously defined compounds elements and amino acids in any combination For example typing a new formula S Combined_ang_brad COOH will add Serine to the previous definition of Combined_ang_brad and add COOH to create a new database entry Introduction 579 580 Chapter 38 Creating a compound database Introduction Compounds can be subtracted from one another in the Formula entry field For example Formula Combined_ang_brad Ser Gly will subtract one Serine
240. e Linear mode This is the laser power at which the data was collected If laser power is adjusted during collection only the end value is stored Displays the mass range over which the ion gate was operating i e defines the parent mass range selected for fragmentation Pulsed extraction was set to on and for a mass of 1050 Da Table 20 6 Graph title information Example item 12mV sum 627mV Profiles 1 54 Customising Graphical Reports Description This displays the value in millivolts of the largest peak in the spectrum This displays the value of the largest peak in the summed traces for shots displayed This displays the profile range of the displayed data Chapter 20 Managing Data Displays Table 20 6 Graph title information Example item Description Smooth Av Specifies that Average Av smoothing was used on this data other options are Gauss Gaussian or Sv GI Savitsky Golay 5 Indicates the smoothing width number of samples constituting a peak used in peak clean up Baseline Indicates that baseline subtraction has been performed on this data 80 Indicates the baseline width used in peak clean up Graph Markers Two types of markers which can be shown on the graphical display are the Baseline and Peak Limits markers Figure 20 33 shows a graph with both of these options enabled The upper trace is the Averaged trace and shows the calculated baseline which will be removed from the pr
241. e On colour green when ready Rotary Always on This pump is used to pump the pump no inlet chamber from atmosphere indication and to pump the exhaust from colours the turbo pumps Turbo pump Fail Off The pumps provide the high Accelerating vacuum They are only or At speed switched off when the instrument is vented The turbo pumps are shown accelerating when the blades are running at less that 80 of full speed Vacuum Fail Poor The gauge is always on The gauge OK gauge reads pressure in Millibar Pascal and or Torr units via a context menu over the window HT supplies Fail Off On The supplies are switched on when the instrument is fully pumped and enabled see Preparation for data collection on page 117 They are always switched off before the door is opened V1 Open This valve isolates turbo pump SAC backing Closed 1 during analyser pumping valve V2 Open This valve isolates turbo pump Flight tube Closed 2 during SAC pumping backing valve Axima Assurance instrument status 95 96 Chapter 11 Checking the instrument status Table 11 1 Axima Assurance status diagram key Item V3 SAC turbo vent valve V5 Gate valve Vacuum state Axima Assurance instrument status Status Open Closed Open Closed Start System Vented Analyser Pumping Roughing Source Source Pumping System Pumped Explanation The valve is shown Open when the SAC turbo pump 1
242. e Peptide Settings window The Display tab of the Peptide Settings The Display tab of the Peptide Settings window Figure 42 6 controls all aspects of the Sequence Calculator view panels It controls the colourmaps font sizes and notation of amino acids within the window The Display tab of the Peptide Settings window is shown in Figure 42 6 below Introduction 605 606 Chapter 42 Sequence Calculator Introduction amp Peptide Settings Tox Keyboard Display Fragmentation Match Peaks Nested PSD Spectra Display Font size E Colourmap Colour Keyboard Short symbol Sequence Short symbol x Report Report mass fe Decimals Include linked sequences IV OK Cancel Figure 42 6 Display tab of the Peptide Settings window Font size The Font size setting controls the size of the font used to display the amino acid sequence in the editor panel There are three options Large Medium and Small The actual size depends on the size of the Sequence Calculator window Choose a smaller size to display more of the sequence and a larger size to see more detail Colourmap A colourmap can be created to adjust the colour of the amino acid classes This is used to display desired properties of the individual amino acids For example using colourmaps it is possible to see at a glance the occurrence of a region of dominant hydrophobicity within the peptide chain Similarly acidic or basi
243. e and the folder and filename can be selected If continuous slides are being used and the current slide is not the last in the series of slides the door will be opened and the sample stage presented for the next slide In this case place the next Slide in the series on the sample stage and either wait for the specified time delay to elapse before the sample stage is automatically retracted or press the Next Slide button to retract the sample stage and continue collecting data Prescan Chapter 14 Collecting data from a sample Automated data quality filtering The Software can be set to automatically filter data during acquisition such that profiles which do not conform to minimum intensity and or resolution criteria are rejected Before a failed spot is rejected a specified number of retries are made in an attempt to eliminate spurious results How the software functions in Auto quality filtering initially depends on whether or not prescanning has been selected A specified number of profiles are acquired from each point in the raster and sorted based on the base peak s maximum intensity A specified cutoff is then used to decide which of the raster points will be used when acquiring a specified number of points Thus for example if 120 points are required and there are 15 raster points which meet the cutoff condition then 8 profiles will be acquired from each point If subsequently at any of these 15 points if all
244. e 20 10 shows a sample spectrum of data The mass range can be increased quickly by using the button in conjunction with the keyboard Pressing the button on the display toolbar with the Shift key doubles the displayed mass range By pressing Ctrl with the range can be expanded by five times and by ten times witn Shift Ctrl as shown in Figure 20 11 Scrolling graphical displays Chapter 20 Managing Data Displays Int 6 7 mV sum 955 mV Profiles 1 142 Smooth Av 20 Baseline 80 wint 6 7 mV sum 955 mV Profiles 1 142 Smooth Av 20 Baseline 80 1534 33 1534 33 100 1535 30 100 Current range X2 80 80 60 60 40 40 20 1536 34 20 o o 1533 1534 1535 1536 1537 1538 1539 1530 1532 1534 1536 1538 1540 1842 Mass Charge Mass Charge Int 6 7 mV sum 955 mV Profiles 1 142 Smooth Av 20 Baseline 80 Sint 6 7 mV sum 955 mV Profiles 1 142 Smooth Av 20 Baseline 80 1534 33 1534 00 c 10 Current range x10 urrent range x5 r 0 Shift Ctrl cad 60 60 40 40 20 20 6 34 o o 1525 1530 1535 1540 1545 1550 1510 1520 1530 1540 1550 1560 Mass Charge Mass Charge Figure 20 11 Increasing the displayed range with the scroll buttons Scrolling graphical displays 351 352 Chapter 20 Managing Data Displays Cursors Vertical cursors can be shown ona display by pressing the mouse ADJ UST button Figure 20 12 Up to two moveable cursors can be displayed J ang
245. e Environment Ee Administrative Tools Element men 7 Settings Shimadzu Biotech Launchpad gt iis Element Database Editor oes Quadralay 2 Oy gt 3 Enzymes Ha A IT RoboHelp Office g 2 a Launchpad ro SmartDraw Help and Support Log Window i SoundMAX BB excess pa 7 Run Startup ae s Symantec Client Security et ES Ein g ample Plate Editor 3 2 Log Off bil volo View Express ge y Turn OFF Computer It is often helpful to be able to get an idea of what elements species may constitute a fragment or mass difference in a mass spectrum Where the fragment is of a relatively small molecular weight an estimate of the elemental composition of the fragment mass may be obtained using the Search window To start the Search window select Search from the MALDI MS programs menu on the Taskbar Figure 39 1 Click the Search icon Launchpad Bia irto Show on startup Iv _Heb 8 b MALDI MS Search Archiver Enzymes SaL Or use the Start menu system Berani Genka reie Figure 39 1 Starting the Search window The Search window takes a list of possible elements or species which the fragment may contain Other types of qualitative analyses may well give an indication as to the species which may be present In the case of synthesised compounds a very good idea as to the nature of the compound will already be available In pe
246. e Name Untted Masse Resolution EAA F F pal N terminus Hydrogen H T Moncistopic I fe C terminus Hydroxy HO x Fragmentation Singly charged x Generate Peak Markers Calculate Cation Proton H x Digest I Length 0 Figure 42 17 Using multiple viewing panels Introduction 617 Chapter 42 Sequence Calculator To delete a panel select the panel to be deleted and choose Delete panel from the Edit menu Each new panel will be displayed with a different colour highlight border The border colour is defined in the Sequence Panel colours window obtained by selecting Options from the View menu Figure 42 18 f default MALDI MS olx File Edit view Instrument Automation Processing Help S Li v Toolbar v Status Bar v Display Toolbar v Basic Parameters Display Contents General Graphs Graph Text Text Report Cursors Peak Labels Options r Acquisition Camera Instrument Status Database Viewer Tile Manager Event Filters Printing Type Colour E Text scaling 100 Margins mm In Left 0 Top 0 Right 0 Bottom 0 Headings Display data and cal Iv Display folder Display instrument IV Display borders V Display title IV Display 1st comment IV Show Auto labels Beak Markers Manual labels Colour Editor button Sequence panel colours P All displays OK Cancel
247. e Target Status LC spot method e Calibration method Method Editor Grothe ipe Auto Experiment f Spot files File Path t4XxXB AR iTRAQ BBE File Experiment Help SWH gt ii O ptio na Reporter lons Experiment Precursor Selection Sample Target Status features and apts Mass PAL ad instrument specific items are in red _ Reference reporter ion X Reporter ion tolerance Da 0 LMZ high mass Da w ChIP Imaging Experiment x This wizard will guide you through the process of setting up a CHIP 1000 imaging experiment Begin a new imaging experiment Open an existing imaging experiment _ ________ Browse Figure 5 12 Automation menus b Automation menu 47 Chapter 5 Window and menu guides Processing menu This menu provides access to windows which govern the processing of collected data clean up the data and analyse collected data using specific techniques Cleaning up data is described in Cleaning up data on page 235 The sequence calculator windows are shown in Sequence Calculator sub windows on page 51 H Calibration SBE Optional featu res a nd ees EEE instrument specific Dataset None i 71 Reference editor Compounds Name toF Le Save i t ems are i n re d Mass Formula Abundance Cursor mass Load named calibration Load Fragment fit 3 Calibration Barent massi 0 0000 th sup
248. e at the nominal value of just over 5x10 mbar After about 1 minute it will be possible to enable the high voltages and so switch to operate once again and use the instrument When the CID gas is switched off it will take a few minutes for the original vacuum pressure to be regained Chapter 13 Preparation for data collection atch IEA A V ed CIE Wod JORO paa Ay Abd m IFAD abs CFR 27d Hue mdame Ao Poat T4 dau Bitto t PDN OIG bb S Buie t FT einer US ped Pred 146 iaaah Damda 2 Dau ap ed paitee n His 2467 1146 Cal 12 teg ath a SED of seat mateuKC tsieu GR V224 Une alogne Jy idb t PE te A jar TT nae Peele 1 ai iaar Aude damire ee S Hiachinge Figure 13 1 CID enhanced immonium ion fragments of angiotensin 2 The Axima Performance uses Helium and a gas inlet port is supplied as standard At the bottom of the window are buttons to control Pumping and venting of the instrument and opening and closing of the door x angio2_msms0006 pi4r_msms0004 MALDI MS ife x File Edit Yiew Instrument Automation Processing Help K ac Display Spectrum Profiles 1 Masses 12 1040 1 gt Optional features and Acquisition instrument specific Maintenance QIT ToF Experiment items are in red Figure 13 2 Instrument menu with Acquisition selected Introduction EE Chapter 13 Preparation for data collection Acquisition foo ae Figure 13 3 Axima Experimenta
249. e available display types and the manual section in which where they are covered Table 17 1 Available display types Display type Heading and page Spectrum Displaying Spectra on page 269 Chromatogram Displaying Chromatograms on page 281 Distribution Displaying isotopic distributions on page 417 Calibration Instrument Calibration on page 459 Reference Displaying reference files on page 423 Polymer analysis Polymer Analysis on page 575 Mass list Producing a table of peaks ina spectrum on page 277 Reference list Listing peaks in a reference file on page 428 Calibrant list Calibration text reports on page 473 Notes Displaying laboratory notes on page 290 Selecting the type of display required 267 268 Chapter 17 Viewing the collected data Table 17 1 Available display types Display type Summary Sequence calculator results Sequence Peak Match Instrument Record Information Auto Experiment Results Peptide Mass Fingerprint Results Mascot Search Results Selecting the type of display required Heading and page Getting a Summary of Run Conditions on page 455 Sequence reports on page 620 Performing a peak match on the loaded sequence on page 638 Summary of sample instrument record information on page 457 Displaying Auto Experiment Results on page 223 Protein peptide analysis using Mascot search engine
250. e data Charged File format Columns 10 33 53 117 133 66 117 176 250 30 Delimiter comma v Decimal places 0 2 289 Export Headings Profile 248 212 171 158 145 143 115 Average Processed Peaks Idefault Averaged data Charged Format Intensity hsi 66 106 234 266 132 234 352 500 604 578 Report intensities as m I p aa 496 424 342 316 290 286 230 default Processed data Charged 119 145 177 211 248 284 317 34 4 362 371 368 355 333 305 274 241 209 default Centroid Peaks data Charged 3092 J0e0006 Profile data Charged 66 124 28 14 11 35 40 46 25 85 51 11 78 130 34 35 10 Joe0006 Averaged data Charged 66 124 28 14 11 35 40 46 25 85 51 11 78 130 34 35 10 J oe0006 Processed data Charged 26 26 25 25 25 25 26 28 29 31 32 33 34 34 33 32 31 Joe0006 Centroid Peaks data Charged Figure 33 6 Example of an export file with all export data selected If the export file is to be written for use on a computer using the UNIX operating system then File format should be set to UNIX rather than to PC Three output options are available for the numerical values which are written to the export file Either Intensity values alone or Mass values alone can be written out or Mass Intensity pairs can also be written out as in Figure 33 7 528 Exporting ASCII data Chapter 33 Exporting data and data displays Export ASCII BBE File format 7 Columns 10 a Delimiter comma x Decimal places 0 a
251. e in the edit boxes located immediately underneath the list box select either Gain or Loss and select the adjacent Insert button The Delete button will remove the currently selected Method Editor 189 190 Chapter 15 Automated operation Method Editor modification entry from the list box and place it in the edit boxes where it can be adjusted and re entered Select the Clear button to remove all entries from the list box Minimum Loss and Maximum gain peaks can be specified respectively as a fraction of or multiple of the primer mass Modification peaks outside these limits will not be reported or considered in the calculations The default for both is 2 0 Beneath the Modifications area of the window are located the other numeric fields which govern the Oligo experiment these are Lower and Upper percentage limits which define the PASS UNCERTAIN FAIL condition for the summed modification peaks area as a percentage of the primer peak area and for the Signal to Noise ratio and Resolution checks on the primer peak The Tolerance entry is used to define a window in one of Daltons milli Daltons parts per thousand or parts per million around each of the expected primer and primer plus or minus modification masses which is searched during peak detection The Signal to Noise ratio used to specify a minimum acceptable signal quality for the peaks can have the noise value estimated in either of two methods One method de
252. e instrument is now ready to perform a basic MS experiment and may be operated using the other controls as described elsewhere in this section Acquiring MS MSn data on Resonance 6l Chapter 14 Collecting data from a sample Table 14 1 Mass ranges of the standard modes Approx peak Button Hee Typical range Low 100 200 Da 100 to 400 Da Low 300 600 Da 250 to 1200 Da Mid 850 1 700 Da 800 to 3 500 Da High 2000 4 000 Da 1 500 to 8 000 Da Hi 3000 5 000 Da 3 000 to 15 000 Da MS mode acquisition MS mode data acquisition is a very powerful feature of the Axima Resonance instrument In this mode of operation the instrument will generate ions from the sample and can then repeatedly isolate precursor ions and fragment them In order to do this the basic mode of operation has to be selected as described in the previous section in order to trap the first precursor mass Next ensure that the MS button in the middle of the laser firing window has been set to On Finally enter the series of precursor ions either using the QIT ToF MS tab of the acquisition window see Setting up MS parameters in the Axima Resonance on page 137 or by using the insert key on the keyboard and then typing in the mass of the precursor ion into the dialogue that will appear Once values have been entered into the MS precursor list they may be deleted using the delete key on the keyboard or modified by double clicking on them in the list with the mouse a
253. e mass axis to expand the mass range lnt 100 35 mV mass 9000 to 11000 Smoothed 1 500 1000 1500 2000 P Profile Figure 17 14 Expanding the mass range on a 3D chromatogram Selecting profile and mass ranges on a 3D chromatogram Using the mouse move the mouse pointer to a position on the graph at the start of the required profile and mass range Press and hold down the mouse SELECT button and drag the mouse diagonally across both the profile and mass axes to the required end point of the range Displaying Chromatograms 287 Chapter 17 Viewing the collected data Release the mouse button and the graph will be redrawn expanding both the profile and mass ranges to those selected with the mouse Figure 17 15 lnt 100 35 mV mass 9000 to 11000 Smoothed 1 Ni Z oS ay EF ae a A Za SX 100 zx ND 504 0 500 1000 1500 2000 Profile Drag the mouse diagonally across both axes lnt 100 37 mV mass 9500 to 10700 Smoothed 1 J 7 sS x 5 z 100 Ss 50 Z 0 as T P 1600 1700 1800 1900 2000 fi Profile Figure 17 15 Simultaneously expanding profile and mass ranges
254. e modes of mass and resolution annotation The Annotation Window Once annotation has been used on a display it can be modified in a number of ways Firstly clicking on an annotation item will display frame handles around its perimeter and the item can then be dragged to a new position on the display Annotation Chapter 20 Managing Data Displays Annotation which contains text will have frame handles on the top and bottom of the grab border as well as on the sides Figure 20 42 Lines and arrows can be extended by grabbing the left or right handle and stretching the annotation to the desired width The font size for text annotation can be increased or decreased by dragging the top or bottom handles to the desired font size OPP ELPLEELEEEPEELE EY LELEEEEEEEEEEEEEE LT PEPLEPLEPLPELOLLPPEOEEELEEEEEEELL 2 Z A 1 53 F 6 a lt A 2 A LOELELELEEEEEELEDELEEEEEEEEEEE ELE Sst eee RR RE ARERR Figure 20 42 Annotation frame handles Double clicking on a label displays the Annotation Window Figure 20 43 and the Annotation Properties window Figure 20 46 These windows can be used to alter the properties of labels or to delete all or selected labels Annotation x Display labels list Type Dataset AN datasets 7 Trace Jan Traces 7 Sort Increasing mass iv angio2_msms0006 0 Processed angio2_msms0006 0 Processed Load Include Save Properties Figure 20 43 Annotation window The A
255. e the space taken up by collected data These options are available on the Storage window Select the tab Storage from the Instrument tabbed dialogue window The Storage window will be displayed and is shown in Figure 14 8 4 Acquisition Firing Exp Tech Auto Quality Storage slide Raster Tuning Average fi profiles Store profiles AV hace Figure 14 8 Storage window Storing collected data Chapter 14 Collecting data from a sample Averaging profiles An averaging feature has been provided which allows a number of single shots single profiles to be averaged together to produce one averaged profile For example to average every ten profiles and store the data after each average would reduce the amount of collected data by up to 90 For one hundred profiles this would store only ten averages a considerable saving in terms of disc space consumption Set Average to the number of profiles to average The MALDI MS software has a built in hardware accumulator which allows the instrument to accumulate data from each shot prior to sending the data back to the computer This results in very high data collection rates Set the Accumulate option to the number of shots to be accumulated within the instrument hardware There are a fixed number of accumulate options of either 2 5 10 20 50 100 or 200 shots per profile Collected peak profiles can be stored for All profiles After average of a number of pro
256. e to an unacceptable signal to noise ratio as the peak ratio conditions were within acceptable limits In addition to the text output file the results can also be viewed in the Experiment overview of the Auto Experiment window where it is possible to toggle between the normal view of the plate and the results of the test see Figure 15 24 below Wells which pass are coloured in green wells which fail are coloured red Method Editor 191 192 Chapter 15 Automated operation and uncertain tests are coloured in grey A diagram of the plate can be printed at the end of the experiment by selecting the Print plate check box A colour printer is recommended for this option Toggle between the normal view of the Experiment plate and a view showing colour coded test results Auto Experiment Check here to print the Experiment plate overview at the end of the run Right clicking gives access to the print plate properties see below Import Ascii text experiment file Plate soe 7Q Q GF Experiment OSBE H4E 790 384 2 8mm wells Selection Experiment Samples Experiment plate overview Selections comprising the highlighted group in the experiment Experiment file name not set Selection Detail Comments and Results Comments Title Comments Prefix I Results File T E mail Notification I Upot completion of this experiment send an e mail to the address specified b
257. eagent used for protein digest Selecting the option None will result in a search of each protein sequence in the database for every sub sequence that matches the other search criteria None is not an option for Parent Mascot searches as enzyme specifically is required Missed Cleavages Specify the maximum number of missed cleavage sites allowed in the search Fixed Modifications Specify any known fixed modifications To select multiple entries hold down the Ctrl or Shift key while selecting each item Fixed modifications are applied universally to every instance of the residue or terminus The search will fail if chemical inconsistent modifications are combined Variable Modifications Specify any known or unknown variable modifications To select multiple entries hold down the Ctrl or Shift key while selecting each item Only 4 variable modifications are allowed variable modifications that apply to termini do not count towards this limit Max Peaks The maximum number of peaks that will be extracted from each spectrum which will then submitted into the search Protein Mass Specify the mass kDa of the protein as a sliding window A protein mass of zero will result in no restriction on the protein mass Peptide Tolerance Specify the error window around the peptide mass Select units between Daltons Da parts per million ppm milli Daltons mDa or percentage Monoisotopic Average Specify monoisotopic or ave
258. eated automatically if required Converts all data files in the current folder beginning with the word test followed by 4 unspecified characters The resultant files will also be located in the current folder and will contain peaks data in mzXML format If any files do not contain processing parameters the current processing parameters will be used No feedback will be given to the user run2xml mzxml pc data test tofparams test run example run hello run Converts test run example run and hello run if they exist in the current directory to raw mzXML using the specified parameter file if old data files are encountered The mzXML files will be placed in the current directory run2xml ic test mzxml process Itest log Converts all data files found in c test to mzXML containing processed data A log file detailing operations will be created in the current directory Using the command line editor Chapter 34 Batch processor XML export Progress and error reporting During the conversion of run files the software will report progress information warnings and errors Where corrective actions can be identified the software will attempt to help you If you use the q command line switch the software will not display any progress information If you use the y command line switch non fatal errors will be suppressed Fatal errors will always be shown Progress Information During processing you will be
259. ect Element Database Editor from the MALDI MS programs menu on the Taskbar Figure 37 4 an Documentation e Archiver 3 Compounds Database Favorites Accessories lt Documents amp Configure Environment mS Administrative Tools amp Element gt Setti Mi Shimadzu Biotech Launchpad gt Pe Anit E Element Database Editor s fan Quadralay gt J Search gt 0 Enzymes t aa RoboHelp Office gt Launchpad o Help and Support a SU Log Window IT Soundmax E3 mavens AE Run Startup eS panes aa Symantec Client Security Sample Plate Editor Log Off bill Yolo View Express PP search Figure 37 4 Starting the Element Database Editor The window shown in Figure 37 5 will be displayed Accessing the database amp Element Database Editor Elements 103 Maze Sort Mass Abundance Chapter 37 Element database Isotopes Abundance Element 6 a gt 1412 98 9 Name Eaton fracas fit Symbol ic af Atomic No e 4 sf tf mnoo A sooo ft mapeo of Melting point sso Aoo pooo Boiling point 4824 foo pooo Specific gravity fo foo pooo Conductivity 0 of Average Mass Total abundance Save Ress 120110369028 100 Figure 37 5 The Element Database Editor window The fields on the window contain the following information Table 37 1 Element Database fields Field Elements Element Name Symbol Atomic number Min DBE Max DB
260. ection popup Multiple sample datasets eooceccene UJ 114 Chapter 12 Introduction to displaying data 1000 In the image above a 384 well Sample plate shows that spectrum from a group of 8 wells have been acquired N22 022 P22 N23 P23 N24 O24 and P24 at the bottom left of the plate Of these wells N22 P22 P23 and O24 are selected for display The spectrum for the multi sample well selection from a single dataset shown in Figure 12 5 above is displayed for the processed traces only in Figure 12 6 below 1 c P23 1 c P22 035 4974 0 1020 1040 1060 1080 11001 2 N22 PXI Figure 12 6 Displaying multiple samples from a single data set The window is now set up to display all four types of spectrum traces Experiment with the settings on the Spectrum Display window to familiarise yourself with the options on the window and their effects upon the displayed data traces Usually the Profile and Averaged traces are of most interest for people wishing to see data as it is being collected The Processed trace is preferred to see the results of data processing such as smoothing baseline subtraction peak centroiding etc after data has been collected These various options will be discussed in detail in later sections Multiple sample datasets Chapter 12 Introduction to displaying data Multisample plate diagrams as shown in Figure 12 5 have a pull right menu with two or three options dependant on
261. ections The Summary report lists the experimental conditions under which data was collected for the currently loaded file s Figure 26 2 shows the first page of a summary report for a single dataset and Figure 26 3 shows the report for multiple datasets The comments for the samples can also be viewed in this display Summaries can be printed as text reports to be attached to the graphical printouts Summary of run wide conditions Chapter 26 Getting a Summary of Run Conditions Data angio2_msms0006 Collected 4 Mar 2005 14 37 Title and Prefix Title Title line text Prefix Prefix text Instrument Shimadzu Biotech Kompact MALDI 6 Software version v2 7 0 Build 20050509 Polarity Positive Flight path Reflectron Laser power 73 Plate 408 wells Samples G7 Profiles averaged 1 Profiles stored At end of sample Gate 1035 00 1060 00 Pulsed Extraction 1050 00 bin 53 Figure 26 2 Summary of run conditions Click on the toolbar page up and page down icons to see other pages in the summary report No Name Date Path Res Power Slide Profiles Averaged Stored Samples 1 arb0024 21 6 97 Ref Low 95 20 sample 200 1 End of sample 2 Argo0005 16 897 Ref Low 47 20 sample 15 1 End of sample 3 bob 22 9 97 Lin High 90 20 sample 1 1 End of sample Figure 26 3 Summary of run conditions for multiple datasets Summary of sample instrument record information The run data file stores instrument information
262. ed fragments species to be marked on the graphs These appear as vertical lines in specific colours with a mass and label displayed These markers make it easy to compare peak positions from collected data with theoretically predicted calculated mass positions These are described in more detail later in Applying peptide PSD fragments as peak markers on page 631 but at this point it is worth noting that if peak markers are to be displayed the options for controlling the appearance of peak markers are shown on the Annotation window Annotation on page 382 Automatic labelling of peaks Peaks can be automatically labelled with their mass values on either all displayed datasets selected datasets or labelling can be switched off Peaks can be also assigned to amino acid differences on the collected data The labelling options are shown on the Display Options window Peak Labels property page Figure 25 6 Labelling peaks 2449 450 Chapter 25 Automatic graph labelling scaling and printing Labelling peaks Display Options BBE General Graphs Graph Text Text Report Cursors Peak Labels Auto generated labels Labet EE Label type Mass x Mass labels 1 Decimal above E ax Added label None ba Angle 0 degrees Bold r Italics z Size so a x10 Underlined I Alldisplays ok _ amy cance Figure 25 6 Peak Labels property page Automatically Generated Label
263. ed trace for the dataset being calibrated The peaks in the collected data must be within a certain tolerance band in order to match the reference peak positions For this reason a value for the Tolerance window must be specified A choice of e Da daltons mDa milli daltons p p t parts per thousand or p p m parts per million is available as units for the tolerance window Figure 27 6 If the peaks in the collected data fall within this window and a suitable match is found the instrument will calibrate up to the highest mass in the reference list Analyte sample masses beyond this calibrated range will be mass assigned by extrapolation The Tolerance window specifies the mass range on either side of the calibrant reference mass within which to search for a calibrant peak Figure 27 7 l 5740 3 Theoretical mass Observed mass a lt Tolerance tolerance 5733 95 p Figure 27 7 Calibration tolerance window Specifying a tolerance of 1 Da will look within a 1 Da window on either side of the reference mass for a peak in the collected data The calibration algorithm fits a straight line through all of the reference points using the method of least squares The position of the line is calculated based on the spread of the reference points Calibration of the instrument Chapter 27 Instrument Calibration If only one calibrant reference is present on the calibrant sample spot then an optio
264. een EJ et al Automatic poisson peak harvesting for high throughput protein identification Electrophoresis 2000 June 21 11 2243 51 Method Formula distribution This feature is part of an optional application Analysing polymers If you have this option please refer to the application guide booklet supplied as part of the option Minimum isotopes specifies the smallest number of isotopes that must contribute to a peak before it is to be considered as a candidate for Poisson modelling to determine a monoisotopic peak mass Peak picking 257 258 Chapter 16 Cleaning up data Peak picking e Maximum intensity variation is a tolerance window which allows the peak intensity to differ from its theoretical value by a specified percentage Candidate peaks which are outside this window are discarded Overlapping distributions is a check box which if selected permits the Poisson modelling to attempt to separate out two overlapping isotopic peaks caused by a sufficient degree of amino acid modification occurring and giving rise to two peaks whose masses are close enough to allow isotopic overlap Minimum peak percent applies only if Overlapping distributions are being considered The algorithm identifies the isotopic masses associated with the dominant monoisotopic mass and subtracts this out of the overlapping distribution if the remaining masses do not constitute at least the specified percentage of the dominant contribution
265. elow pelx Type Stat End Samples Mpthod Dafa File Cmts Enter a title and prefix to be associated with all data in the experiment Check the box and enter a filename if a results file is to be generated Figure 15 24 Auto Experiment window Method Editor Chapter 15 Automated operation Email notification Tick this box and enter the required email address to receive an automatic email when the experiment has finished If you receive an error message the Email feature is not available If you require this feature 1 Close this window 2 Open a MAPI client e g MS Outlook not Outlook Express 3 Open this window and the email feature is available Print plate properties Options available for text header information for the printed plate are set in the Print Properties window Available from a Pull right menu on the print plate icon Click right mouse button Plate woe 7 QQ f Print Plate Properties Experiment filename Raw Data Filenamess Overall test statistics T Date and Time J Experiment comment T Cancel Figure 15 25 Starting the Print Plate Properties window Progress After you have started an experiment you can view its progress in the top right corner of the Auto Experiment window E3 1 Parent acquire E3 1 Acquisition Starting E3 1 Parent acquire data AE0003_0039 E3 1 Acquiring data AE0003_ 0039 E3 1 Laser shot 48 of 100
266. els can be hidden from view by selecting the labels to be hidden and pressing Hide selected labels or Hide all labels to hide all labels in the list A hidden label will appear in the list with an asterisk in front of the label name to indicate that the label is hidden Labels can be restored to the display shown again by selecting Show selected labels or Show all labels To delete a specific range of labels place a pair of range cursors on the graph encompassing the labels to be deleted and select Delete listed between cursors To add new labels in the selected display click on New the New Annotation window will be displayed allowing new labels to be added Annotation labels can be saved to named files which can be copied and archived for later use To save a Set of labels click on Save the Save as window will be displayed Figure 20 45 Chapter 20 Managing Data Displays Type in a file name for the new labels file the file extension will be lab and press Save Save As 21x Save in O labels amp ek Ge File name PO Save as type Label files lab Caca Figure 20 45 Save labels window To load labels which were saved previously click on Load the Open window will be displayed Select the labels file to load and press Open Note that loading a set of labels will remove any labels currently defined on the display and replace them with those from the selected file To include labels
267. elves to be exported so that applications such as Microsoft Word or Microsoft PowerPoint can paste them directly into reports The two options available are to either export the entire window as a meta file or export a selected tile as a metafile The exported file is actually an enhanced metafile structure which allows the image to be moved around resized and also to be edited by the importing application Exporting data displays as meta files Chapter 33 Exporting data and data displays Exporting mz data formats You can export the spectural data shown in the current MALDI MS main window as either mzxXML file mzData file From these data formats you can use third party software to view your data perform protein peptide analysis etc If you wish to export bulk data see Batch processor XML export on page 537 mzXML file The mzXML format is an XML eXtensible Markup Language based common file format The supported version of mzXML is 2 1 For more information visit the Seattle Proteome Center SPC Proteomics Tools NHLBI Proteomics Center at the Institute for Systems Biology web site http www proteomecenter org Exporting to an mzXML file 1 Display the sample profiles and mass range of the data which you wish to export 2 Select File gt Export gt mzXML File Processed data file Peak data file Lok carve Exporting mz data formats 531 Chapter 33 Expor
268. ent reference file e g as P14R_ peaks Do this before trying the fragment fit because any peaks which are not found in any data set will be discarded and have to be re typed 14 Set the search tolerance to 500mDa 0 5Da 15 Select fit fragment calibration in the fragment calibration setup window The results of the fragment calibration will be displayed as the number of times each peak is used and the average error of the peak When complete check that 492 Axima Confidence Chapter 28 Fragment ion calibration All of the fragment peaks listed in the setup window have been used in some of the fragment spectra no completely missed peaks The residuals are no more than 0 2Da ideally they will be lt 0 1Da 16 As a check if fragment fit is ticked in the calibration window the mono isotopic mass of all the fragments peaks in any of the 10 spectra will be within 0 3Da of their theoretical value If the fragment calibration procedure has failed any of the fragments in the list or failed completely check for the following common mistakes t If the The search tolerance was not set to 500mDa half a Dalton The apparent masses are the actual fragment masses and vice versa The sample is not P14R residuals are more than 0 2Da then possible reasons are the wrong apparent mass has been assigned to that fragment the peaks for that fragment in more than one calibration spectrum are systematically high or low
269. ent status The Axima instrument status diagram gives an overview of the instrument status in real time Instrument Status _ m gt Figure 11 6 Axima Resonance status diagram The key at the bottom of the diagram indicates the possible states of the unit Axima Resonance instrument status ececcece o te Chapter 11 Checking the instrument status Axima Resonance status diagrams key The items on the diagram and their possible states are described in the table below Table 11 3 Axima Resonance status diagram key Item Laser Rotary pump Turbo pump T1 T2 T3 T4 Vacuum gauges HT supplies 104 Axima Resonance instrument status Status Off On Always on no indication colours Fail Off Accelerating or At speed Fail Poor OK Fail Off On Explanation The laser is shown in the Off colour blue when not ready to fire and in the On colour green when ready This pump is used to pump the inlet chamber from atmosphere and to pump the exhaust from the turbo pumps The backing line gauge pressure is shown The gauge reads pressure in Millibar Pascal and or Torr units via a context menu over the window The pumps provide the high vacuum They are only switched off when the instrument is vented The turbo pumps are shown accelerating when the blades are running at less that 80 of full speed The gauge is always on T
270. ent window on display in the main window is printed Select After Average to print after acquired profiles have been averaged together Select At End of Sample to print at the end of sample acquisition Select whether to print in Monochrome or Colour in the Printing type box The font size when printing can be defined in the Text Scaling field This is scaled as a factor to the current font size on the display hence 100 prints fonts same size as display Set the printed documents margin dimensions in the Margins section of the window Method Editor 197 198 Chapter 15 Automated operation Export The Method Editor Export window is identical to that described in Exporting ASCII data on page 524 To access the Export window select the Export label from the tree as shown in Figure 15 28 below Method Editor default mtd Of x Sample Method Sample Method default mtd Parent Data Export iy Parent El MY Acquisition M RA Raster Export ASCII text x VX Auto Quality M 3 Laser Firing File format PC Columns 10 4 VE Processing VILE Calibration Delimiter comma x Decimal places 0 a EZ Peak Cleanup Mile Monoisotopic Peak Picker Export Headings oe VEA Peak Filtering Average Processed Peaks VIS Mascot I Applications i amp Compare Sequence Format Intensity 7 Oligo Analysis 4 Output YQ Print J Report intensities as my I I Report ie lon Finder OE MS MS Sy Acquisition PE
271. ents in the elemental formula must be in the element database Defining protecting groups Protecting groups are used to attach to amino acid Sugar sequences to protect the sequence from cleavage by enzymatic digests or other reagents The Compounds Database can define these protecting groups Chapter 38 Creating a compound database Protecting groups may be defined within the Compound Database by selecting Protecting Group as the Category and clicking on the New button The Edit Compound window will be displayed with the Protecting Group property page shown as in Figure 38 6 Edit Compounds General Compound Amino Acid Sugar Protecting Group Terminal Group Cation Nucleotide Name FO Symbol __ Formula CY Replace Modes Figure 38 6 Edit Compound window for Protecting groups Type in a Name and Formula for the protecting group The group can have a Symbol which will be displayed in the Sequence Calculator window as an attachment to the main sequence e g Boc for t Butoxycarbonyl The Replace entry should contain the elemental composition of the group which is replaced by the protecting group In most cases this will be a single hydrogen atom but this could be another elemental species If this protecting group usually only modifies certain specific amino acids or sugars then the single letter mnemonic for this specific site should be entered here A warning will be issued if the protecting gr
272. equencing Results Terminus N Direction TE Match tol 0 4 Discount immonium ions T Generate enerate OK Cancel Figure 42 30 Nested PSD tab of the Peptide Settings window In the Subtract File Parameters area enter suitable mass range tolerance and threshold values The mass range ensures that noise outside the range of the peptide will not be included in the analysis and the threshold should be used to select valid peaks from within this mass range The threshold value differs from the peak cleanup threshold value in that it is the minimum apex intensity for a peak to be included in the analysis The tolerance is the maximum mass difference between peaks in different files for them to be matched Tick the Show spectra check box if the results of subtraction are to graphically displayed Note that Peaks should be displayed in the Display contents window Using the Add file button select the data to be subtracted then press the Subtract files button Note that the data processed during this operation is also governed by the parameter settings in the Peak cleanup window Introduction 635 636 Chapter 42 Sequence Calculator The software will now determine the order of file subtraction by comparing the centres of the ion gate used in the acquisition of each spectrum and then perform the subtraction of files starting with the two highest mass gate centres For each subtraction the software will generate 3 files that h
273. er Parent 1722 958 Da gill 4532616 LGDVVLGFDSVDPYVK Hit 1 2 3 14 516 M 1722 9583 14 9 9 99 The first three fields allow you to personalise the subsequent report as shown in the example above 1 In the Name Email address and Report title fields enter the information you wish to appear at the top of the Mascot Search Results page 2 At the Top hits to report field select the required number from the drop down list 3 If you want to include the Mascot Overview Table feature in your results tick the Produce an overview section tick box Mascot MS MS searches 305 306 Chapter 18 Protein peptide analysis using Mascot search engine Protein identification search criteria Protein identitication search criteria Ro Enter any known details about the sample and its preparation Taxonomy Arabidopsis thaliana thale cress Digest enzyme Trypsin v Missed cleavages Fixed modifications Amidated Protein C term Ammonia loss N term C Carboxyvmethyl C Variable modifications Acetyl K Acetyl N term Acetyl Protein N term Amidated C term Amidated Protein C term Ammonia loss N term C Biotin K Biotin N term zl Protein mass 0 kDa Treat masses as Monoisotopic Average Peptide tolerance 0 5 Peptide tolerance unit Da het Decoy In the Taxonomy field select the required source of the protein In the Digest enzyme field select the required enzyme and in the M
274. er and is controlled by a software application called MALDI MS The software also processes the resulting data and displays it on the PC monitor The Axima is used for scientific research and quality control analysis Skills This guide assumes that you are familiar with using PCs and using the Windows XP Vista operating system Comments While we always try to ensure that the content of our publications is accurate we know that mistakes can be made In a continuous attempt to improve we welcome details of technical inaccuracy and any comments on the content and format of the publication Please email comments to tech pubs kratos co uk Screen shots The screen shots used in this guide are for guidance only It is not practical to cater for every eventuality and therefore the best representation is used For clarity the screen shots are taken from a Windows XP operating system set to the Windows Classic theme About this User guide 10 Chapter 1 Introduction Using the mouse The conventions for using the mouse assume that the buttons and centre wheel on the mouse use the default settings Typical affects with MALDI MS software Left mouse button Selects buttons check boxes etc Centre mouse wheel Applies cursors to a spectrum Right mouse button Displays a menu If they have been changed for example for use with the left hand adapt the instructions in this guide to suit Disclaimer and copyright The informa
275. ererreres 238 S CON AMO siidcnrewiadianedan sxaadhe abeataldcwasndaadkeabuxeauus a a 239 Smoothing collected data cece cece e cette teeta eee ene ed 240 Subtracting the baseline ceece cece eee eee tenet eee eee need 243 Peak detecti M rrai iiite Piwes cadt E a a 247 Peak Reporting ws siccsacnnssncueaws saanciaimeceeomseennetas naina 251 Double threshold iessisircnnisiiricnnsrinnni a ai 254 Peak picking iieri inoia annann aaa AARE A APN EAR ts 256 Filtering specified peaks 20 0 0 cece cece cece e eee e neta eta eee ened 259 Viewing the collected data ccccceeeceeeeeeeeeeeeeeeeeeeeeeeeeuseeenseeans 265 LMtrOCUCHION secs cvadecee setae ec etees enna enira kaea i a cee es 266 Selecting the type of display required ccceceeeee eee ee eee 267 Displaying Spectra cc cece cece eee eee ee eaten neta eats teeta eee 269 Producing a table of peaks in a SPECtrUM cceceee cence sees eaes 277 Displaying Chromatograms cceceeee ee eee eee eee eee eee ed 281 Displaying laboratory notes cece cece eee eee eee eee teeta eed 290 Protein peptide analysis using Mascot search engine 293 PMEPOGUCHION ve deeiemsades E 294 Accessing the internet search form cceceeee ee eee ee eee tees 295 Mascot PMF Searches ecceceee eee eee e teen e estes ta eee teeta ees 296 Mascot MS MS Searches ccee eee e eect eee ee teeta e estan ta eee
276. erminate MALDI MS was unable to allocate a new timer either close down other running programs or restart Windows to free the available timers Error 10010 Operation failed Could not open the clipboard Restart Windows Error 10020 Could not paste clipboard contents Check to see whether the Windows clipboard is operative and working properly by opening another Windows application and trying to cut and paste Otherwise restart Windows Error 11000 Unable to calculate distribution The Windows event log may contain more specific descriptions of the errors encountered possibly in sufficient memory space Error messages Chapter 44 Summary of error messages Error 12000 Cannot open file for writing filename Retry the operation check file and directory write permissions on the directory being written to Error 12010 Cannot open the import file filename for reading Check that the file filename exists in the selected directory Error 12020 Error reading from database The Windows event log may contain more specific descriptions of the errors encountered check that the database exists and that its file size is not zero Error 12030 Exceeded maximum number of panels sequence not loaded A maximum of 10 panels is permitted delete a panel or more to load the new sequence Error 12040 Cannot seek to end of file filename Error appending the end of the file check that the file is a valid file and is non zero in length Error
277. es If the instrument has had micro biological substances bacteria viruses etc inserted then 1 Were any of the substances living or active yes no 2 If yes what class of active micro organism Class 1 yes no Class 3 yes no has been processed Class 2 yes no Class 4 yes no Note If the instrument has had living micro biological substances of class 2 inserted then the instrument must be de contaminated before it is either returned to Kratos or before a Kratos engineer is asked to work on any contaminated part of the equipment There must be a signed declaration that the decontamination has been carried out to approved procedures these must be referenced and copies supplied to Kratos on request If the instrument has had living class 3 or 4 micro biological substances inserted then the instrument must be serviced by trained engineers of the institute or organisation to which the instrument belongs If you have any doubts about the classification of the substances that have been processed through the instrument then please contact Kratos Where there are any doubts then the action taken must be commensurate with the highest classification that may have been in contact with or processed through the instrument ii Bio chemical Samples Soluble in aqueous Soluble in organic detergent solutions solvents such as Please categorise the samples as follows acetonitrile a biologically active proteins peptid
278. es 141 Setting the laser POWED cece eect eee eee teeta eee eee eee ed 145 Storing collected data ccceceee cece eect ee eee etna eae eae ee ened 146 Averaging profiles 2 0 cc ceseeeceeee cis ce bb ae ence cteea oie eek na ceed cade 147 Firing the laser cece eee eee eee eee een eee 150 Automated data quality filtering eceeee cece ee eee eee 153 Using the camera 1 eect eee eee eee eet e eae eene ed 157 Blanking low mass iONS ceceee ee eee eee eee eee eee 159 Acquiring MS MSn data on Resonance cceeceeeeeeeeeeeeen ees 161 Automated Operation cccccececeeeeeeeeeeeeeeeeeeeeeeeeaeeeueeeeeaesaeeanenens 165 PMUPOGUCEION EEEE ea roe E E Guage baeteneseecets Saeed ens tend 166 Method Edit auscissecitncesencsawedas Sasaenaleie ace seradddmanedtennedvecatings 167 Auto Experiment sssrini erien a iE 203 ASCII text experiment file formats esseesseeeeserrrrerrrrrerns 210 ASCII Text Method file format cece cece cette teen eee ed 214 Displaying Auto Experiment Results ceeeeeeee ee eee eee ee es 223 Using Auto Experiment Results VieWer cceceeeee eee ee tees 225 References 0 0 cece eee ene ene eee e eee ne tate 233 Cleaning Up data cccccceeeeceeeeeeeeeeeeeeeeeeuaeeeeaeeeeaeeaeaeeeuaeeaeansenas 235 I MUPOGUCHION ves oevtecedeas ste eee a ri E i aa Eaa aini DE 236 Combining tagged profiles sssssssssesssssrrsserrresrrrrr
279. es turbo pump SAC backing Closed 1 during analyser pumping valve V2 Open This valve isolates turbo pumps Flight tube Closed 2 and 3 during SAC pumping backing valve Axima Performance instrument status 101 Chapter 11 Checking the instrument status Table 11 2 Axima Performance status diagram key Continued Item V3 SAC turbo vent valve V4 CID purge valve V5 Gate valve V6 CID valve Vacuum state 102 Axima Performance instrument status Status Open Closed Open Closed Open Closed Open Closed Start System Vented Analyser Pumping Roughing Source Source Pumping System Pumped Explanation This manual valve is shown Open when the SAC turbo pump 1 is vented The valve is shown Open when the CID gas lines are purging Isolates flight tube from analyser for door opening Shown as open when instrument is acquiring Allows CID gas to enter the flight tube Instrument just switched on System is vented at atmosphere for maintenance Pumping down Flight tube Begin to pump down the source Pumping down SAC only Instrument is pumping ready for data collection Note The vacuum system shows System pumped when this pumping starts The system will not be ready to collect data until one minute after the turbo pump is up to speed and the vacuum gauge reads 2x10 gt Chapter 11 Checking the instrument status Axima Resonance instrum
280. es yes no yes no yes 02 b synthetic proteins peptides yes no yes no yes no c nucleic proteins peptides yes no yes no yes no d synthetic compounds yes 2 yes no yes no iii All Bio chemical Polymer or other Samples Dangerous to Human Health You must declare the nature of the hazard to health taking account of the maximum amount and variety of sample processed and all their associated risks Comments a Toxic yes no b Immunogenic yes tN c Corrosive yes OD d Reactive yes no iv Radio Active Contamination If the instrument has had radio active substances inserted or has been in a radioactive environment then the following questions must be answered 1 Has the instrument been decontaminated to an approved procedure yes no Please quote procedure reference 2 Has the instrument been tested after any decontamination for A es no residual radio active levels y If yes what maximum radiation level was recorded Note An Instrument that has been radio actively contaminated must not be returned to Kratos unless it has been fully de contaminated and is in a safe condition Now please complete sections 4 and 5 on side 1 DOC 075 page 2 2 Issue7 October 2005 Chapter 3 Getting started Chapter 3 Getting started 24 Chapter 3 Getting started Introduction Introduction It is assumed that the user is conversant with the normal opera
281. ethods is outside the scope of this manual but adequate information is available from the original references 7 HPLC Colour coding is performed based on the Browne Bennett and Solomon HPLC index and is an indication of the retention of the peptide on a reversed phase HPLC column Although absolute values are not always meaningful they do give an idea of the relative retention time and elution order within a group of peptides being separated by liquid Ul Introduction 607 Chapter 42 Sequence Calculator chromatography In general the lower the index the shorter the expected retention time on the column See reference 1 for more information The BB HPLC HW KD and ESG options are coded using a grey scale Black represents a minimum value and white a maximum value Keyboard The Keyboard option controls the notation used in the Keyboard tab see section on page 604 This can be set to either Short Long or Full notation Sequence notation The sequence displayed in the view window can be shown in either a Short symbol or Long symbol notation Examples of both views are shown in Figure 42 7 Sequence Calculator ojx File Edit View Settings a i PPPPPPPPPPPPPPR Short symbol notation e gt gt gt I Synchronise Name Unte Masse Resolution a aj N terminus Hydrogen H 7 Monoisotopic 7 24 C terminus Hydroxy HO 7 Fragmentation Sinay charged 7 Generate Peak Markers Calcu
282. ew menu Or click on the toolbar display contents button The Spectrum Contents window will appear Figure 12 2 pil4r_msms0004 MALDI MS OF x File Edit view Instrument Automation Processing _ Help ola v ee Display Spectrum v Profiles 1 Masses 12 1040 1 gt v Status Bar v Display Toolbar v Basic Parameters a4 The display contents toolbar button Camera Spectrum Contents Instrument Status Database Viewer Tile Manager Event Filters Dataset 1 ang11__0001 Scroll ai Traces Profile Average Process Peaks View Stack Overlay Set 15 610 Display multiple samples Figure 12 2 Spectrum Contents window 3 Select Sets 1 5 to display the first 5 datasets up to 10 can be displayed 4 Make sure that the ion trace charged ions as opposed to neutral fragments is displayed by selecting the J Trace option 5 Select the same dataset for processing by selecting the BA Process option 6 Select Profile Average Processed and Peaks Displaying spectrum seses ry D Chapter 12 Introduction to displaying data 7 Select an Overlaid view traces drawn on top of one another rather than the isometric projection view when Stacked is selected 8 Press the Apply button to update the display An example of the four types of spectrum displays is shown below 980 mY Profile 54 Profile 980 mv sum 52915 mW Profiles 1 5
283. ex Data Acquisition Data Acquisition CID on 968 52 a C Program Files Shimadzu Biotech Launchpad Data Customers University of St Andrews 417 4E0023_000 Results Start End Result 16 08 2006 14 28 50 16 08 2006 14 30 37 Completed Data Acquisition CID on 1251 74 Data Acquisition CID on 909 02 Data Acquisition CID on 1618 91 For Help press F1 Compress tree nodes To compress a node move the mouse pointer over the node and double click Adjust column widths You can adjust the width of a column 1 Moving the mouse over a column headers divider until the resize column pointer is displayed b 2 Hold down the left mouse button and move the mouse to resize the column Displaying mass lists For Mascot Search Investigation nodes you can view the mass lists submitted to the Mascot search engine 1 Double click the required node to reveal the Data File node Using Auto Experiment Results viewer 227 228 Chapter 15 Automated operation 2 Double click the empty node to reveal the mass list File Edit View Instrument Automation Processing Help Sjaj e 2 a a4 Display auto Experiment Resuts Profiles 1 16 Masses 1459 2426 IPI am Files Shimadzu Biotech Launchpad Data Customers University of St Andrews 417 auto experiment MS_MS rex Data Acquisition CID on 1251 Data Acquisition CID on 909 02 CID on 925 04
284. example myinputwell txt which describes the individual wells The file consists of one line per well Each line must be as follows WelllD CentreX CentreY Type Dimension1 Dimension2 where e WelllD is the well identifier e g A1 CentreX is the horizontal distance mm from the left hand edge of the plate to the centre of the well Sample plates and plt files Chapter 13 Preparation for data collection CentreY is the vertical distance mm from the bottom edge of the plate to the centre of the well Type is either C S or R for Circular Square or Rectangular Dimension1 is the diameter if the well is circular well otherwise it is the width both in mm Dimension2 is the height of a rectangular well mm The terms left hand edge and lower edge are as indicated in Figure 13 8 above i e defined with respect to the entry of the plate into the sample chamber of the instrument Individual line items must be separated by white space white space being a TAB or space character So for example the file myinputwell txt shown here describes two circular wells Al and A2 both 2 5 mm in diameter at different plate locations Al 70 0 120 0 C 2 5 A2 65 0 120 0 C 2 5 B1 70 0 115 0 S 2 5 C1 70 0 110 0 R 2 5 2 0 A 2 5 mm square well B1 at a third location and a rectangular well C1 of width 2 5 mm and height 2 0 mm at a forth location To convert this file into a normal plate file say fourwell plt start up an MS
285. f TOF2_mixture pos_ref E TOF2_mixture_external pos_ref E TOF2_mixture_internal pos_ref amp My Documents PI My Computer kia amp nmi File name TOF2_misture pos_tef 7 Places Files of type any x Cancel Figure 27 5 Reference files list This list gives the name of the reference file and the ionisation polarity with which the file can be used Loading a new reference file will over write the currently loaded reference file and any changes or amendments which may have been made to the currently loaded file will be lost Make sure that any changes are saved before loading a new reference file The loaded reference file may be displayed as a graph or a text report See Displaying reference files on page 423 for details Loading a reference file Chapter 27 Instrument Calibration Calibration of the instrument A calibrant reference file should ideally be created with the calibrant reference masses of all expected references which are on the calibrant sample spot Load this calibrant reference file by pressing the List References button on the Calibration window and select a reference file from the list Figure 27 5 Check that the masses displayed in the list are those required Any reference masses not expected in the calibrant sample can be deleted using the Delete button Creating a new reference file on page 463 If a calibrant reference file has not been created a
286. f profiles selected However selecting twenty segments would split the mass range chosen into twenty equal divisions and draw twenty traces one for each division Set Segments to the number of segments required in the chromatogram display In this instance a three dimensional chromatogram plot of a sample is shown giving the variation of intensity with profiles and mass Figure 17 11 Displaying Chromatograms 283 284 Chapter 17 Viewing the collected data lnt 100 2 0 mY mass 3715 to 14680 5000 10000 Mass Charge Figure 17 11 Three dimensional chromatogram display In the example above it can easily be seen that the peak giving the largest intensity m z 10 000 is concentrated in profiles 1600 1900 There are distinct regions of interest appearing in different ranges of profiles at different masses These regions appear as a contour map within the data collected allowing regions of interest to be quickly recognised More data can then be collected from the regions of interest Where a mixture of peaks of interest of varying molecular weights are present this type of display can be of particular use In the examples given the chromatogram is used to show signal variation from profile to profile showing the range of profiles at the front face of the 3D plot and the mass range going diagonally back into the picture It is also possible to produce a 3D image with the mass scale shown at the front and the
287. f sample 7 Figure 15 5 Method Editor Data storage Method Editor 169 Chapter 15 Automated operation Raster The Method Editor Raster window is identical to that described in Defining a sample raster for acquisition on page 132 To access the Raster window select the Raster label from the tree as shown in Figure 15 6 Method Editor default mtd Oix Sample Method Sample Method default mtd MS MS Rastering Yq Parent Vy Acquisition v fa Raster OO ZX Auto Quality Sae v 3 Laser Firing MEQ Processing Raster Type Regular Rectangular VL Calibration Peak Cleanup E Monoisotopic Peak Picker VEA Peak Filtering Width 96 00 um Height 116 00 um VSR Mascot l I Applications Centre x joo um Centre Y po um Compare Sequence ie ae PS oi Oligo Analysis 0 000 um ha Wy Output foes Ap Print Calculate Raster Based On Spacing IV Raster Style 3 Export Use Parent Points r I Report ie lon Finder 7A MS MS EME Acquisition Peak Selection v Ea Raster v 3 Laser Firing VY Processing EZ Peak Cleanup ZI Monoisotopic Peak Picker VEA Peak Filtering VSR Mascot My Output X K Get Manual Settings Save Method Load Method Figure 15 6 Method Editor Raster The method editor version of rastering allows the points obtained in the Parent pre scan to be used in the MS MS rastering This is done by selecting the Use Parent Points option from the MS MS Rastering dialo
288. ferent instrument modes e g a simulated resolution of 400 would produce peaks approximately as they would appear in linear mode and a 1000 resolution would simulate reflectron mode Figure 23 7 shows an example of the theoretical distribution for Polyethylene glycol H O CH z CH gt 35 H O CH3 CH gt 56 Figure 23 8 shows an example of four reference compounds used to provide peaks at different masses Reference peg_ref Int Resolution 1000 at 50 100 Profiles 30 60 40 20 0 100 80 60 40 20 0 2000 Mass Charge Figure 23 7 Example of PEG theoretical distribution Displaying reference files Chapter 23 Displaying simulated data Int Resolution 1000 at 50 147 1 377 3 100 600 800 Mass Charge Formula m z Cs H9NO4 147 1317 C22H35N0O4 377 5284 C44H69N13011 956 1214 APGDRIYVHPF 1253 4377 Peptide sequence Figure 23 8 Example of reference display for four reference peaks Reference displays can be expanded in the same way that spectrum and chromatogram displays can Simply drag the mouse with the SELECT button held down across the region of interest Figure 23 9 shows an expanded region Displaying reference files 425 Chapter 23 Displaying simulated data Reference peg_ref Int Resolution 1000 at 50 100 80 60 40 20 0 Reference peg_ref Int
289. files or At end of sample data collection When set to never data is never stored It must be noted that in order to be able to reprocess the scans at a later date profile data must be stored This can be either single profiles or an average of a number of profiles but the profiles must be stored To be able to look at individual profiles the profile data must be stored for every profile collected Otherwise the only option available is to look at the average of a number of profiles Where Store profiles has been set to Never and some interesting data has been seen press the Store button on the Laser firing window to allow the data collected to be saved to disc for future reference Data compression A form of data compression is already incorporated in the MALDI MS software to reduce the amount of data written to disc when data is stored This removes repeated data samples having the same value and stores only the value and a count of the number Averaging profiles 47 148 Chapter 14 Collecting data from a sample of repeated values Thus 100 occurrences of a sample data value of zero only takes up 2 data values saving 98 repeated values This can significantly reduce the size of a data file In addition to the form of data compression just described previous versions of the software allowed all of the data files collected to be compressed further on storage at this release the extra compression option has been removed from
290. fines noise as the median of all of the intensity values in the tolerance window which do not contribute to the peak If unusually the limits of the peak extend beyond the tolerance window then the window is extended by the tolerance value beyond the peak limit for the purpose of calculating the noise Alternatively the noise can be the estimated baseline value at the peak apex This is the method used in the S N values reported in the Mass List display We define the signal as the peak apex intensity The only other entries on the Oligo Analysis window govern the output of results Select the Spectrum META file check box if you want to output a spectrum of each sample Using the Mass range display limits such spectra can be extended to separately specified percentages of the detected peak range at either end of the plot both default to twenty percent of the range beyond the minimum loss peak mass and the primer peak mass of each sample The META files will have the same base name as the results file but will have the sample well ID and sample source identification code tagged on separated with underscore characters _ along with the emf extension The Results text file is specified in the edit box at the bottom of the window again a Browse button is provided for navigating the file system The results file if it already exists will be Chapter 15 Automated operation appended to unless the Overwrite existing file check box is
291. fix field Select from the drop down list to either apply the comments to the Current dataset or to the Next data collected Chapter 10 Putting comments with collected data 3 Click the Apply button your comments appear on the spectrum If the second line of comments does not appear you need to change the setting of the Display Options window a From the MALDI MS window select View gt Display Contents b If required click the General tab c Tick the Display 1st comment tick box Display Options BEI General Graphs Graph Text Text Report Cursors Peak Labels r Acquisition Display After Average v Print or x r Printing Type Colour x Text scaling 100 Margins m In C Left jo fo Right Bottom jo r Headings i Display data and cal IV Display folder Display instrument IV Display borders IV Display title IV Display 1st comment IV Show Auto labels Peak Markers Manual labels Sequence panel colours T All displays d Click the Apply button Clearing comments 1 Click the Clear button 2 Click the Apply button Copying comments from other datasets To copy comments from another loaded dataset select the dataset from the list of loaded datasets adjacent to the Copy comments from gt button and then press Copy comments from gt the comments will be copied into the window Apply the comments to either the currently loaded data
292. for each individual sample acquired These records can be viewed by selecting Instrument Record I nformation from the Display options and then selecting the Display Contents either from the View menu or from the toolbar button A report such as that shown in Figure 26 4 below is produced Summary of run wide conditions 457 Chapter 26 Getting a Summary of Run Conditions Data lt Untitled gt E3 c ep 4 A g R Shimadzu Biotech Axima Performance 2 8 0 20070831 Mode Reflectron Power 90 Source chamber vacuum pressure Pa 1 565 Flight tube vacuum pressure Pa 1 3E 5 CID cell vacuum pressure Pa 8 3E 6 Laser power 90 Laser repeat rate Hz 10 000 Source HT voltage V 19941 404 Lens HT voltage V 6520 180 Pulsed Extraction HT voltage V 2494 698 X source deflector voltage V 0 351 Y source deflector voltage V 1 041 Gate positive voltage V 0 000 Gate negative voltage V 0 244 Linear HT voltage V 0 000 Xreflectron deflector voltage V 59 482 Y reflectron deflector voltage V 0 259 Reflectron HT voltage 7 V 24336 004 LMZ off LMZ mass Da 0 000 CID off Figure 26 4 Instrument Record Information The display contents window shown below can be used to toggle through any of the samples from which data has been acquired Also any of the other data sets currently loaded can be selected from the Dataset combo box in this window W Instrument Record Listing BBE Dataset E 1
293. g The laser power defined during rastering can be altered as a percentage of the parent laser power by defining the percentage increase from the Laser Power Change field 170 Method Editor Auto Quality Chapter 15 Automated operation The Method Editor Auto Quality window is identical to that described in Automated data quality filtering on page 153 To access the Auto Quality window select the Auto Quality label from the tree as shown in Figure 15 7 Method Editor default mtd Sample Method ME E Sample Method default mtd Wy Parent Eh MY Acquisition v Raster Auto Quality 3 Laser Firing TE Processing M Calibration EZ Peak Cleanup IME Monoisotopic Peak Picker MIX Peak Filtering MS Mascot OS Applications amp Compare Sequence O Oligo Analysis My Output M Print O Export OW Report J a lon Finder I MS MS MEI Acquisition BE Peak Selection M Raster M 3 Laser Firing MEI Processing EZ Peak Cleanup MILA Monoisotopic Peak Picker MEA Peak Filtering ME Mascot yy Output X gt Data Storage Get Manual Settings Parent Auto Quality m Monitor Mass range 50010000 ie Start noise 5000 Noise width 50 q Auto power Power limits jos tl Start power jo a r Criteria Min intensity fis 4 mY Min S N E a Min S N po 500 Min resolution z Max rejects 5 Z r Auto aim Prescan V j1 Profiles Pt Cutoff feo 4
294. g box is of a standard file open selection type but only files that exist are allowed to be selected Data File An editable field with a dialog box activation button that contains the data file that the mass spectral data will be written to The dialog box is of a standard file open selection type files that do not exist are permitted Comments A read only field with a dialog box activation button This is used to enter or modify the comments which is a user settable free text field for each of the samples in the group The information displayed in the field is the number of samples with a comment of length greater than zero to the total number of samples in the group The dialog box contains a list control with two columns Well ID and Comment The Well ID field is the location of the sample and is read only and Comment is the comment for the sample and is an editable field Auto Experiment 209 210 Chapter 15 Automated operation ASCII text experiment file formats The 2 types of ASCII experiment files which can be imported into the Auto experiment are described here The only essential difference between the two is that the first assumes that a standard plate is defined and loaded see Experiment Controls on page 206 so that all well locations are predefined the second defines its own plate by specifying the location and size of samples and so is particularly suited for 2D gel experiments Both file formats a
295. ging Data Displays Chapter 20 Managing Data Displays 333 Chapter 20 Managing Data Displays Introduction The data displays show both graphical and text reports of data These displays can show data collected from the instrument calibrant reference files isotope distributions in general any type of data supported by the Launchpad suite of software The data displays are shown within the main MALDI MS window Clicking the mouse MENU button right mouse button within the display area of the MALDI MS base window shows the Display menu This menu is used to create and manipulate displays Gr Gon Paste Gut Sopy Past Column Inert Delete P Rom Colm Annotate Inset gt Annotate Annotation PIGBEES notato Properties Inset Tags Tags gt AllInsets Peak labelling Peak labelling gt New Annotation Cixi Ammotatomnkroperies Tags Peak labelling 334 Introduction Decimals fno X o angio2_msmst Chapter 20 Managing Data Displays Annotation Properties Se x Paste Type Insert VY Text 4 Delete r Data Properties Annotate Dataset Mass trom Trace to p Display Properties Font Arial s Bold D B Italics Size fao 10 Underline Angle 0 Sj degress Colour Set Transparent C Opaque ay tect Annotation Properties Tags b Peak labelling
296. ging sequence calculator COIOUIS cece e eset teeta 440 Automatic graph labelling scaling and printing csscsesesees 441 I MEPOGDUCEION de musiecen couse sian e e weve ent eenes 442 General display Options ccceecee cece ence eee eee eee teeta eatin 443 Markers displayed On a Spectrum 0c cece cece eee ee eee eee ees 445 Labelling Peaks creiser aan o agence tens 449 Getting a Summary of Run Conditions ssssssssssnsnnnnnnnnnnnnnnnnnnn 455 Summary of run wide conditions sssssssssesssersrererresererrrsres 456 Instrument Calibration ssssssssssnnnnnnnnnunnnnnnnnnnnnnnnnnnnnnnnnnnnnn na 459 Kaldgololira di elnie E E ined eiaeeegaies 460 Creating a new reference file u sssssssssssrrrrsrrrrrerrrrrrerrrrres 463 Loading a reference fil 0 cc cece ee cece eee eee teeta eee ee teeta eee 466 Calibration of the instrument c eee ee cece eee eee ee eee ee eee 467 Nonlinearity COrrectiOn ccc cece ee eee eee eee eee eta teen rerent 480 Fragment ion Calibration ccccceeeceeeeeeeeeeeeeeeeeeeeeeaeeeeoeeeeaeaeees 481 N rOdUC ION secs cine dey cnteeee ences ikan cee eete ye keen anda come biG aE 482 Axima Performance ccccece eee ee eee eee eee eee a a tate aE 483 Axima Confidence cris secenene eceeyeety ce en eak 489 lon gate Calibration ccccceeseeeceeeeeeeeeeeeeeeeeeeeeeeeeeeeuaeeeeueeeoneneee 495 LMEFOGDUCHION A A E A E TE 496 Check
297. h short lived samples to leave printing and updating the displays until data collection has been completed Displaying Spectra Chapter 17 Viewing the collected data Producing a table of peaks in a spectrum Set the Display to Mass list to see a text report of peaks found in a spectrum The report has the following columns Table 17 3 Columns in mass list Column Meaning Mass The mass of the peak centroid or apex Area The area under each peak as a percentage of the largest peak Total The area under each peak as a percentage of the total peak areas Apex mV The largest signal value in the peak Resolution M dM at 50 peak height M mass Signal Noise Apex signal divided by the base line noise Flags U Unresolved S Significant M Manually assigned I Monoisotopic A Most abundant F formula distribution Unresolved significant and manually assigned peaks are displayed in a different colour on the peaks trace the colour of which can be set on the Colour chooser window Producing a table of peaks in a spectrum 277 278 Chapter 17 Viewing the collected data The contents of the mass list report are controlled by the Display contents window for mass lists Figure 17 7 2 Mass List Contents 1 x Dataset Trace Sample Total Resolution Signal Noise Flags Precision 2 decimals Maximum listed peaks 50 Minimum peak apex 0 000 x il Significant peaks only I
298. h the ion gate on Axima Performance 485 Chapter 28 Fragment ion calibration 9 The fragment calibration setup window is accessed from the calibration window by selecting set up in the fragment calibration mass section mid right hand side of the window H Calibration Bik Calibrant references r Calibration files lt Untitled gt J11 List references Reference editor Compounds Name tof List Save Formula Abundance Cursor mass Time Load named calibration x Load 190 05 H 1 E Calibration Fragment Correction _ x Apparent mass Actual mass Formula Fit error Da Reference filename P14R_TOF2 frag_ret Save it through zero Sav Apparent mass 458 8448 m Insert Delete Clear list tt Actual mass 195 1100 Formula Tol 500 mDa 7 F Combined Cal Calibration parent mass 1533 8580 fal Average vy 71000 Fit fragment calibration a 10 The fragment reference file P14R_TOF2 can be loaded from the setup window Alternatively if no reference file is available type in the actual and apparent masses given in table above 11 Ensure that the parent mass is set to 1533 86Da actual and apparent 12 For each fragment peak in the list a select the peak by double clicking on the entry in the list b enter the apparent mass the mass without the fragment calibration applied of the fragment peak in the P14R
299. h to perform the search You can add parent peaks to the search list where each parent entry contains a list of MS MS peaks The Add Parent button fetches peaks from the currently loaded spectrum whereas Add File s allows you to fetch peaks from many data files at once You can then search Mascot directly using this list or save the list to a Generic Mascot file for use elsewhere Parent and gP 1722 958 Parent 1722 958 Da Fragment list for MS MS search 5 Double click the title to reveal the mass list Mass list This section allows you to specify the list of masses on which to perform the search You can add parent peaks to the search list where each parent entry contains a list of MS MS peaks The Add Parent button fetches peaks from the currently loaded spectrum whereas Add File s allows you to fetch peaks from many data files at once You can then search Mascot directly using this list or save the list to a Generic Mascot File for use elsewhere Parent and Fragment list for MS MS search 28 246 503 eo 261 128 eo 261 363 eB 261 754 o 286 378 oe 357 505 2 357 859 358 777 eB 385 441 8 385 776 2 484 651 8 504 676 xl Save List as Clear List Delete Peak Add File s Add Parent 6 You can delete a peak select it and click the Delete Peak button Adding files You can search using the results from several experiments For example you could sa
300. have an alias other name by which it will also be recognised in any formulae This is accomplished by typing a colon in the Name entry followed by the alias name The amino acids have been entered in this manner e g Arginine Arg and R are all aliases for arginine and can be used in any formulae Any number of aliases can be created for each compound entry For example Formula Arginine Arg R Asparganine Asn N Aspartic_acid Asp D Introduction 573 574 Chapter 38 Creating a compound database Introduction Defining sugars Sugars may be defined within the compound database by selecting Sugar as the Category and clicking on the New button The Edit Compound window will be displayed with the Sugar property page shown as in Figure 38 4 Edit Compounds General Compound Amino Acid Sugar Protecting Group Terminal Group Cation Nucleotide Name Formula Figure 38 4 Edit Compound window for Sugars Type in a name and a formula for the new sugar definition into the Name and Formula entries respectively The same general rules apply for sugar definitions as for general compounds Where a sugar definition is used in a compound definition for another database entry the sugar must be placed within braces to avoid confusion with another definition which may have the same name i e where hex could be hexane or hexose then hex unambiguously denotes hex to be a sugar rather than a general com
301. he gauge reads pressure in Millibar Pascal and or Torr units via a context menu over the window The supplies are switched on when the instrument is fully pumped and enabled see Preparation for data collection on page 117 They are always switched off before the door is opened Chapter 11 Checking the instrument status Table 11 3 Axima Resonance status diagram key Item Inlet Valves Vacuum state Status OK warm hot Open Closed Start System Vented Analyser Pumping Roughing Source Source Pumping System Pumped Explanation Readback of the ambient air temperature sensor The valves open close accordingly Instrument just switched on System is vented at atmosphere for maintenance Pumping down Flight tube Begin to pump down the source Pumping down SAC only Instrument is pumping ready for data collection Note The vacuum system shows System pumped when this pumping starts The system will not be ready to collect data until one minute after the turbo pump is up to speed and the vacuum gauge reads 2x10 gt Axima Resonance instrument status 05 Chapter 11 Checking the instrument status 106 Axima Resonance instrument status Chapter 12 Introduction to displaying data Chapter 12 Introduction to displaying data 107 108 For Help press F1 Chapter 12 Introduction to displaying data Introduction The data collected by t
302. he on off switch at the back of the instrument If the fault persists call service Error 24000 No data to process Error 24010 No data to process Error 24020 No data to process There is no data currently loaded therefore the processing requested has failed Error messages Chapter 44 Summary of error messages Error 24030 Calibration failed Check that there are reference peaks within the specified tolerance Error 24030 Cannot open the run file to store the comments Retry after checking file and directory write permissions on the databases directory being written to Check the available disk space in the directory being used Error 24040 The filename run file already exists Use another filename or delete the existing filename Error 24050 Cannot open the run file to store the run statistics Retry after checking file and directory write permissions on the databases directory being written to Check the available disk space in the directory being used Error 24060 Cannot allocate memory for manual peak assignments Error 24070 Insufficient memory available Error 24080 Memory not available for creation of a raw buffer Error 24090 Memory not available for creation of an averaging buffer Error 24100 Memory not available for creation of a background data buffer Error 24110 Memory not available for creation of a centroided scans buffer Error 24120 Memory not available for creation of a processed scans buffer
303. he reference file generated by the Polymers window Select whether Profile or Peaks or both displays are required If viewing profiles set the resolution at which the display should be simulated and select Auto mass range Press Apply to create the polymer reference display Figure 40 5 Introduction 593 594 Chapter 40 Polymer simulation Reference nylon Int Resolution 2000 at 50 2038 12 1925 45 2151 28 2264 75 1699 30 Figure 40 5 Reference file generated by the Polymer window Saving a polymer series Having created a polymer series the parameters used to generate the series can be saved as a named file for use at a future date To save a polymer file simply type the name of the file into the Polymer files entry and press Save When the file is re loaded the polymer series will be regenerated Introduction Chapter 40 Polymer simulation Loading a polymer series A previously stored polymer parameter file can be loaded by pressing the Load button A list of saved polymer files will be displayed Figure 40 6 Polymer Files 21 x Lookin gt Poymes sti EE nylon6 polymer es test polymer Files of type Polymer files x Cancel Figure 40 6 Polymer Files window Select the file to load from the list using the mouse SELECT button the file name will appear in the Filename entry Press Open to open the polymer file and re generate the serie
304. he Do Not Print list Items can be removed from the Print list by selecting the item and pressing the button Similarly items can be added to the Print list by selecting the item and pressing the 4 button Properties for each item can be accessed by selecting the relevant item and clicking the Properties button as shown in Figure 15 27 Chapter 15 Automated operation pectra Mass List Sequence Report n Spectra Properties xi Window ee Report Properties x Print blank line after Section Headings T Print instrument name V Print Column Headings IV prope xl Print Blank line after Column Headings Print comments title JV Print folder name No properties for this selection Print borders IV A Lines to print es a men Print 1st comment IV cms Mass List Properties x Mass List Report Settings Mass List Contents Settings Print Section Headings IV Mass Area l Print blank line after Section Headings Total Apex m Resolution Signal Noise Print Column Headings IV Flags Precision 2 decimals Maximum listed peaks 20 EEE e a Minimum peak apex o 000 x Il i Significant peaks only I Print Blank line after Column Headings M Cancel Figure 15 27 Properties windows for auto printing Table 15 3 Spectra Properties Print data and Print the dataset filename and the calibration calibration filename name P
305. he file may have been corrupted An error occurred writing to the ASCII export file Retry the export check file and directory write permissions on the directory being written to Sample number was not acquired Data was not acquired for the sample number requested Unable to read elemental database The elemental database file may not be present in the databases folder or the file may have been corrupted Insufficient memory available Close other programs shutdown active processes to free more memory Error messages 649 650 Chapter 44 Summary of error messages Error 4020 Error 4030 Error 4040 Error 4050 Error 4060 Error 4070 Error 4080 Error 4090 Error 4100 Error 4110 Error 4120 Error messages Invalid formula cannot be converted formula The formula contains invalid unknown symbols which cannot be parsed into a meaningful formula Please recheck your formula see Rules for entering formulae on page 573 Unable to allocate memory for formula to mass conversion Close other programs shutdown active processes to free more memory Unable to allocate memory for reference entries Close other programs shutdown active processes to free more memory Cannot have zero time squared coefficient The calibration is invalid use another calibration or revert to a factory calibration original calibration Error writing to reference file filename occurred while operation Retry the opera
306. he instrument is shown in the display area of the base window There are many types of data displays which will be covered in detail in Viewing the collected data on page 265 and Managing Data Displays on page 333 Only a brief summary of the main features are given here which allow viewing of data collected In order to familiarise yourself with the data displays and spectrum traces load the example data which is shipped with the MALDI MS software suite installation This data is found in the datat efault folder and is called default Load the example data following the instructions given under Loading data on page 75 J ang1i__0001 MALDI MS lolx File Edit view Instrument Automation Processing Help sal Aee Sl 2 E display Spectrum Profiles 79 Masses 1041 1053 gt Data ang11__0001 021 3 Feb 2005 11 20 Cal jo_cal 3 Feb 2005 11 01 Kratos PC Axima CFRplus V2 4 0 Mode reflectron Power 64 Blanked P Ext 2300 bin 138 lnt 655 mV Profile 79 100 Display toolbar se 60 40 20 o 398 mV sum 11941 m Profiles 50 79 Smooth Av 20 Baseline 80 1046 5 1047 1048 1049 1050 1051 1052 1053 Mass Charge Status bar displays any status messages Figure 12 1 MALDI MS base window Introduction Chapter 12 Introduction to displaying data Displaying spectrum To see the available displays make the following selections 1 Set Display to Spectrum 2 Select Display contents from the Vi
307. he ion gate 499 Chapter 29 lon gate calibration Calibrating the ion gate There is an ion gate calibration for each of the Mass Range buttons positive and negative modes You only need to calibrate the ion gate for the mass ranges and modes that apply to your experiments Each calibration is a two point calibration Reset the ion gate calibration IMPORTANT You must reset the ion gate calibration to avoid calibrating on top of a previous calibration 1 In the QIT ToF MS window click the Adjust Gate button Quick Gate Adjustment x m Mode Mass range mid e50 x Polarity Positive x m Parameters Point Point2 Set mass 1000 Da 2000 Da Actual mass 1000 Da 2000 Da Results Gradient Offset 2 Click the Reset button and check that the gradient is 1 and the offset is 0 Calibrating the ion gate Chapter 29 lon gate calibration Acquiring the calibration parameters 1 Using the table below as a guide identify the approximate lower and upper calibration masses Calibration range Button Lower mass Upper mass Low 100 150 350 Da Low 300 300 1 000 Da Mid 850 1 000 2 500 Da High 2000 2 000 5 000 Da Hi 3000 3 000 10 000 Da 2 Using the procedure described above Checking the ion gate on page 497 identify the mass set in the Resolution field of the QIT ToF MS window e the actual mid point of the ion gate Setting the calibration 1 In the QIT ToF
308. he likelihood of finding a match e g green monkey as a keyword will produce significantly less matches than just searching for monkey Be as specific as possible whenever specific information is available use this information to restrict the search Usually peptide chains have an attached adduct or cation e g H Any cation selected on the Sequence Calculator window see for example Figure 42 2 on page 602 will be added to the database entry mass shown in the Load Sequence window list of matching sequences Terminal groups are assumed to be attached to all sequences in the database The terminal groups selected on the Sequence calculator window will be added to the database entry sequence mass shown in the Load Sequence window list of matching sequences Press the List button and the selected database will be scanned for matching entries Any matches will be listed in the scrolling list depending on the size of the database and the search parameters specified this search can take a while to complete The list displays three columns Keywords Mass and Sequence name The keyword score is calculated by adding 1 point for every keyword matched If the user specifies a Sequence then only database entries which contain this sequence are listed though the status information at the bottom left of the window indicates if other entries were encountered which matched some of the keywords but not the sequence The list is sorted with hig
309. hecking file and directory write permissions on the directory being written to or disk space Error 16300 Unable to reserve memory for periodic table data Error 17000 Insufficient memory to create RGB table Close other programs shutdown active processes to free more memory 654 Error messages Chapter 44 Summary of error messages Error 17010 Cannot open RGB colour table filename Colour conversion will not be performed Check that the file filename exists in the selected directory Error 17020 Insufficient memory to create RGB table entries Close other programs shutdown active processes to free more memory Error 18000 Cannot read compounds database Check that the file compounds data exists in the databases directory Error 18010 Cannot read compounds database version Error 18020 Compounds database invalid version Error 18030 Cannot read number of compounds in database Retry loading the file the file may have been corrupted or may contain invalid information If this is the case copy the original compounds data file into the databases directory or restore a previous version from a backup Error 18040 Unknown compound group read assuming general A compound was read from the database but the group was unknown it has been assigned to the general category The file may be very old and will need recreating Error 18050 Cannot update compounds database Error 18060 Cannot create compounds database Retry afte
310. hest scoring entries first Mass is the molecular weight of the database entry sequence including terminal groups and cations if selected Sequence name is the database entry sequence name Chapter 42 Sequence Calculator Double click the mouse SELECT button on the peptide sequence that you wish to load from the database the selected entry will be highlighted If a sequence already exists in the selected panel a popup warning message will be displayed Figure 42 11 A sequence is loaded in the selected panel Do you wish to overwrite the sequence Figure 42 11 Overwrite warning popup The option will be given to either overwrite or insert into the current sequence in the selected panel with the newly selected entry from the database Selecting Insert will insert the selected sequence at the current cursor position s within the loaded sequence In this manner long sequences can be constructed from numerous different database entries Editing the sequences Many of the features of the editor will be self evident to the regular computer user aS most are standard text editing functions Essentially the sequence in the viewing panel is treated as a simple text string and is handled in a word processor like fashion The sequence is edited using the Edit menu which appears when Edit is selected on the toolbar Figure 42 12 Sequence Calculator Of x File Edit View Settings Paste Ctrl Find and Repla
311. hods of peak detection and reporting with parameters Option Threshold Apex Apex peak 1046 56 _ Threshold Start End Threshold Centroid Centroid peak 1046 51 Equal area _ _ Threshold Start Gradient Centroid Centroid peak 1046 67 Equal area Start Explanation Peak is calculated as the highest point above the threshold Peak is calculated using the area between the curve crossing the threshold Peak is calculated using the area between the curve crossing a channel Chapter 16 Cleaning up data Table 16 1 Methods of peak detection and reporting with parameters Option Explanation Threshold 25 Centroid Peak is calculated Equal using the area Centroid peak 1046 54 top 75 used between the curve crossing the threshold Only the top 75 of area is used to _ _ Threshold calculate the peak Start End Gradient 25 Centroid Peak is calculated i Equal area using the area Centroid peak 1046 55 top 75 used between the curve crossing a channel Only the top 75 of area is used to calculate the peak Start End To set the same peak clean up parameters on all displays set the Apply to gt option to All displays Having made the relevant selections for the data processing parameters press the Apply to gt button to apply the changes Peak Reporting 253 Chapter 16 Cleaning up data Double threshold Double threshold thi
312. ialog displayed depends on the field selected There are four types of fields in the list control read only editable editable with dialog box and editable only with dialog box The fields are described as follows Type A read only field that indicates whether the samples in the group are mass scale calibration samples Cal or processing samples Pro This information is derived from the method being used for the sample group Cal samples are identified on the sample plate by an orange ring around the selected well If Cal group s is are preceding Pro group s sample processing order is optimised so that Pro samples are associated to the nearest Cal sample and that nearest Cal sample is processed prior to the associated Pro samples In this case samples may be processed outside of the normally expected sample group running order Start An editable field that is used to denote the location of the first sample in the sample group This is normally set using the Selection plate and wg or buttons End An editable fiela tnat is used to denote the location of the last sample in the sample group This is normally set using the Selection plate and faq or buttons Auto Experiment Chapter 15 Automated operation Samples A read only field that denotes the number of samples in the group Method An editable field with a dialog box activation button that contains the method file to be used to process the sample group The dialo
313. ication Environment Configuration Editor Environment Variables Mascot Setup Launchpad files Home JE Program Files Shimadzu Biotech Data C Program Files Shimadzu Biotech Comments C Program Files Shimadzu Biotech Reference C Program Files Shimadzu Biotech Calibration C Program Files Shimadzu Biotech Export C Program Files Shimadzu Biotech Peptides C Program Files Shimadzu Biotech Polymers C Program Files Shimadzu Biotech Databases C Program Files Shimadzu Biotech Labels C Program Files Shimadzu Biotech Parameters C Program Files Shimadzu Biotech Internet C Program Files Shimadzu Biotech Stage files JC Program Files Shimadzu Biotech Method C Program Fies Shimadau Biotech Filters C Program Files Shimadzu Biotech Experiment C Program Files Shimadzu Biotech Log C Program Files Shimadzu Biotech I Save parameters on exit Print Settings Reset Figure 6 1 Environment Configuration Editor variables N au kh W Click in the required path typically C Program Files Shimadzu Biotech Select a new drive from the Drive list or expand the tree Select a folder or subdirectory from the tree Press the Set path button Repeat this procedure for each path to be changed and then press Save the changes will be written to the Windows registry and will be used throughout the Launchpad software suite Environment Configuration Editor 6l 62 Chapter
314. ick the Apply button 3 Open the Firing window and locate the fullerite sample left of the P23 on a 384 well plate 4 Acquire a spectrum and view the mass range around the 1000 Da peaks lt Untitled gt MALDI MS File Edit View Instrument Automation Processing Help sla Ea 2 S x a4 display Spectrum z Profiles 1 Masses 941 1054 14 gt 379 m sum 75843 mV Profiles 1 200 Unsmoothed 984 00 985 00 1010 1020 1030 1040 NYZ dM 4 62 M dM 216 31 NUM A Checking the ion gate 497 Chapter 29 lon gate calibration 5 Zoom in on area between two prominent peaks it is easier to see the boundaries of the ion gate when there are no prominent peaks to dominate adjacent peaks lt Untitled gt MALDI MS File Edit View Instrument Automation Processing Help slal AES 2 S x a Display spectrum z Profiles 1 Masses p87 1007 14 gt lnt 41 m sum 8196 mY Profiles 1 200 Unsmoothed 987 02 100 1000 1002 1004 1006 11c P For Help press F1 dM 4 62 M dM 216 31 Num Z 6 Note a peak close to the area of interest in this example 1000 90 Da 7 Click the Abort button 8 Go to the QIT ToF MS window Acquisition positive Firing Exp Tech Auto Quality Storage slide Raster Tuning QIT ToF Ms 2 3 MS MS Precursor Ion 999 980 Ei Precursor Ion 0 000 Ei Resolution Std Isotopic Selection
315. ide mid Places Save as type Text Files txt 7 Cancel Figure 19 8 Creating a results file 2 Navigate to the required folder and type in the filename for your results file 326 Defining destination of results file Chapter 19 lon finder 3 Select the Save button the folder and filename information appear in the lon Finder window jolt Finder x Ion search template Settings Add entry Paste data Import data Remove entries Remove all Results file destination Folder C Documents and Settings Adr Folder file Filename 7 peptide mix txt I Auto select filename Dialogda Load new Save Process Figure 19 9 lon Finder window results file details Auto select filename The filename will be based upon the spectrum name or date if no name is specified and Sample plate well reference s 1 In the lon Finder window tick the Auto select filename checkbox 2 Select the Folder file button Browse For Folder MEG Select a results destination Folder Desktop E B My Documents El My Computer E3 o My Network Places a Recycle Bin O mass lists Make New Folder Figure 19 10 Browse for folder window 3 Navigate to the required folder Defining destination of results file 327 Chapter 19 lon finder 4 Select the OK button the folder and path appear in the lon Finder window jolt Finder x m Ion sea
316. ides In other words the presence of such fragments is seldom of any use in identification of a particular peptide Automatic peak selection Type the mass range in the Masses entry over which peak selection is to be performed The number of peaks to select is specified by the Peaks option Press List peaks to create the list of peaks from the chosen mass range Cursors can be used to select a range of masses from a spectrum display Position two cursors bracketing the mass range of interest and press the m button Masses will be set to the range marked by the cursors Introduction 637 638 Chapter 42 Sequence Calculator Manual peak selection Peak masses can be entered manually by typing in the peak mass in the Mass entry and pressing the Insert button Cursors can also be used to select a mass from a spectrum display Position a cursor on the spectrum at the mass required and press the peL button Mass will be set to the peak mass under the cursor To delete a mass from the list click the mouse SELECT button on the mass to be deleted and press the Delete button To delete masses in the list select either All masses or Selected mass and press Delete gt Peaks can be searched in the currently loaded sequence in databases on the local computer hard disc or network drives and on remote databases via email Performing a peak match on the loaded sequence In order to perform peak matching with the loaded peptide sequence selec
317. ies with display types v Toolbar v Status Bar T aldas K Les v Display Toolbar v Basic Parameters Display Contents Options Camera Instrument Status Database Viewer Tile Manager Events Logged t Camera Settings Brightness 59 Contrast 61 C oe Hue N A Saturation N A M Vertical Mirror fil H i Zoom 130 5 M Scale down to fit 4 H gt F Show Scale Click right A Position x 3 Position Y 20 Pe ft en a T mouse button Size 53 I Alignment marks 4 gt Live I Move cross hairs Move alignment marks Freeze Copy Image BWCCIR BWRSI70 PAL NTSC Save Image OK Apply Cancel Exit Figure 5 5 View menu a View menu Chapter 5 Window and menu guides 9 Instrument Status oj x Vacuum state System pumped ips Gauge 2 Tuning mode reflectron_ms Lisi Instrument mode Reflectron cD Ro ar V4 Purge l ve Gauge 4 3 7E 7 mbar Gauge 1 4 3E 6 mbar Door closed Pump Supplies Pressure Valves Atspee On Ok Open bw pnm Accelerating Poor B Y motor Off Off Closed cistern Fail Fail Fail B Inlet Status window for Performance Compound Database Viewer L x Toolbar v Status Bar v Display Toolbar v Basic Parameters Display Contents Options
318. iew is restored to its original state The group of arrow key buttons is used to move the X Y sample stage bringing any particular point into focus The central block filled button stops motion of the stage Am 4 Two other buttons exist only on the Axima instruments Firing Tab these are the raster button S which when selected causes the XY stage position for laser firing during an acquisition to be governed by the raster currently loaded in the Raster Tab dialogue window If no raster is specified during acquisition then the laser is fired at the centre of selected wells The x manual control button gives the user full control of the position on the plate that the laser is fired at Navigation can be achieved either by use of the arrow buttons or the Goto location pull right menu option Presently a well must be selected for manual control to operate but this is only to give the sample a name e g A1 and in no way restricts the location of laser firing to the specified well if a raster is specified in the Raster tab dialogue then this will be applied about the manually selected location rather than the selected well Sample selection Chapter 14 Collecting data from a sample Setting the laser power The power of the laser is controlled using the Power setting which can be used to select any power the range 0 180 where 180 represents full laser power This is achieved by rotating a graduated density wheel in the path
319. indow provides tab access to the menus which control collection of data from the samples and govern how and when data is stored See Collecting data from a sample on page 139 for details Fring Exp Tech Auto Qualty Storage side Raster Tunno I Mto quality m o a ae aa ei Protes E per sanle Shots acmm sted per protte r Pulsed Datraction optimised at Da te wa X t mee e r Qa Accumiote Profiies to Fio Acquiing tangle G14 Fring Exp Tech Auto Quotty Storage Side Roster Tuning Tuning mode z bosa Sit _ coma Mass Range 1 0 100000 Max Laser Rep Rate 100 QD _ Enable Acquisition default_linear Fring Exp Tech Auto Qualy Simango Sise Renter Turing Merece Aito power Mass range ME tt Bower fits 3040 tL Sra notte 0 Hoe wae Saree Cienia Min intensity 15 mi Min Se 5 pena f 4 ProfiewPt Minimcckdion 500 3 Maswa o H Mnn Maxton gt 2 X Cupes 20 4 I Auto Qualy Data I Prescan Osta I Overte Data View Data fuso calitxation Lock Mars Calibrator MT Lock Mass L ie fT i Pailonsanos marcueeneed options C Abert eoperment Prompt for um s imtervertion Skip to rmm samgle Ce Firing Ep Tech auto Qualty Storage side Raster Tuning Average T Z potis Soomes SE Fring Exp Tech Auto Quotty Storage Side Roster Tunng Sample plate fle arva Plate Carrier Edt sang
320. ing distributions My Output Minimum peak percent Vi Print I Export Q Report a lon Finder vI MS MS MI Acquisition SE Peak Selection v 333 Raster 3 Laser Firing VI Processing EZ Peak Cleanup A Monoisotopic Peak Picker VILE Peak Filtering VS Mascot My Output X Data Storage Get Manual Settings Save Method Load Method Figure 15 13 Method Editor Monoisotopic Peak Picker The monoisotopic peak picking parameters can be setup as detailed in Peak picking on page 256 Method Editor 177 Chapter 15 Automated operation Peak Filtering The Method Editor Peak Filtering window provides the functionality to define filters to ignore certain mass spectral peaks To access the Peak Filtering window select the Peak Filtering label from the tree as shown in Figure 15 14 below Method Editor default mtd L 1 x Sample Method Sample Method default mtd Yi Parent El MY Acquisition M fa Raster vix Auto Quality Peak Filter Editor V3 Laser Firing VEY Processing VL Calibration E Peak Cleanup MIL Monoisotopic Peak Picker Parent Peak Filtering vS Mascot J Applications amp Compare Sequence Oligo Analysis M Output VE Print 3 Export i I Report Selected Filter ie lon Finder k Append Delete Clear vE MS MS Dee date _ Le M Acquisition SE Peak Selection VIER Raster Tolerance 0 5 a Da bd M 38 Laser Firing S AER Processing Convert formulae a
321. ing the ion gate ssssssssssesssssresssrrrrrsrrrrrerrrrrrererrnnes 497 Calibrating the ion gate ssssssssssssrrrssrrrrrsrrrrrnrrrrrrsrrrrrres 500 Chromatography cccceceeeeceeeeceeeeeeeeeeeeeeeeeeeaeeeeeeeeeaeeeeeeeeeeneeees 503 UFO CU CHIONN iiss ciass pe tre Ficce ctessccta srev e worlaiate i vivladle vle EE 504 Chromatographic peak detection c cece eee ee ee eee eee eae 505 Starting another data processing WINKOW scseseeeeeeeeeeeneeees 513 Archiving data wscciiedeiscisedaaceeseretaeteantindeeeateetedaediedennnedeieenetwe 515 LMEPOGUCHION meiir o e ie a e i eE 516 Exporting data and data displayS s ssssssssssnssnnnnnnnnnnnnnnnnnnnnnnnn 523 Exporting ASCII data scscsvsciicirairisnnisen nak 524 Exporting data displays as meta files cc eene 530 Exporting mz data formats cece cece teeta eee ee teenie 531 lon FINGER 2 edenndier eid hheke wed meets idnomeetieberstendslaeomenererboes 534 BIOM AD aicsiacisintsshantcaldeieatewiles E ANNEDE Pa sauna DENRA EN SEAN D EAAS 535 Batch processor XML export sssssssssnsnsnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 537 IMEFOGUCHION eissir tenrai Eaa R A 538 Using the Batch processor sssssssssssssssirssssirrererirnnnernr rnern 539 Using the command line editor ccc eee e eee eect eee ee eee es 547 Using the clipboard cccsceceeeeceeeeeeeeeeeeeeeueeeeeeaeaeeeuaeeeeaeeouenees 557 Printing the contents of displayS
322. int is shown in terms of its rank according to the summed intensity of signal it produces Automated data quality filtering Chapter 14 Collecting data from a sample Using the camera The Axima instruments ship with a built in camera optionally The camera can prove useful with some samples in choosing sweet spots by inspection To turn on the camera display select Camera from the View menu options see Figure 14 13 below J p14r_msms0004 MALDI MS Oy x Fie Edit view Instrument Automation Processing Help Le z v Toolbar 2 Display Spectrum X Profiles 1 X Masses 12 1040 1 gt I eee V Status Bar v Display Toolbar v Basic Parameters fi Camera Viewer Display Contents Options Camera Instrument Status Database Viewer Tile Manager Event Filters amp Camera Settings Brightness 59 Contrast 61 a a E a Ue a Click right mouse A mE N A z a N A a button M Scale down to fit rE ri gt I Show Scale Position x 3 Position Y 20 Freeze M Cross hairs aj E gt Ki C gt Copy Image Save Image Size 53 M Alignment marks al E gt Exit n IV Move cross hais M Move alignment marks BW CCIR BW R5170 PAL NTSC OK Figure 14 13 Using the ARDIA ARNET to inspect a sample we Using the camera a N 158 Chapter 14 Collecting data from a sample The camera is normally set so that the cross hairs are at the centre of the image and
323. io2_msms0006 MALDI MS of x File Edit Yiew Instrument Automation Processing Help i y Se 2 8 x a Display Spectrum Profiles fi Masses 400 638 l gt I r Data angio2_msms0006 G7 c 4 Mar 2005 14 37 Cal tof 4 Mar 2005 14 37 CID of 1046 51 Shimadzu Biotech Kompact MALDI 6 V2 7 0 Beta 1 Mode reflectron_ms_ms Power 73 Gate 1035 00 1060 00 P Ext 1050 bin 53 lnt 3 7 m sum 199 m Profiles 1 54 Smooth Av 20 508 33 Cursor mass shown in footer vog Cursor 543 34 566 29 520 540 560 580 600 Mass Charge Mass 533 88 Closest peak M dM 195 93 Figure 20 12 Single cursor on a display When a single cursor is shown the value under the cursor either mass or profile is reported in the bottom left hand corner of the window In the case of mass being displayed the resolution M dM at 50 peak height of the closest peak to the cursor is also displayed Mass cursors are shown by default on spectra and profile cursors on chromatograms the cursor reflects the units shown on the display graph s x axis Cursors Chapter 20 Managing Data Displays To position a mass cursor e Click and hold down the mouse ADJ UST button anywhere on the graphs Move the vertical cursor to the desired position on the graph so that it is directly over the mass or position of interest and release the mouse button When both cursors are shown the range between the cursors is reported in the bottom right hand
324. ion M H C6 H15 N4 02 175 21 Bull Breese 690 00 z 175 16 HPLC 3 60 175 12 H IR Elemental composition M H Average mass M H Most abundant mass M H Monoisotopic mass M H C terminus Hydroxy HO C12 H26 NS 03 288 37 Bull Breese 760 00 288 32 HPLC 3 00 288 20 Singly charged Fragments Average Masses M H n a 17 b 17 o sass 1 69 16 2 225 35 97 17 253 36 Figure 42 24 Peptide report generated for the peptide Dynorphin 627 628 Chapter 42 Sequence Calculator Introduction Modifying the terminal groups Select the N and C terminal groups of the peptide using the N terminus and C terminus entries on the Sequence Calculator main window The blank entry at the top of the list can be used to simulate cyclic peptides which have no terminal groups Similarly select the cation from the list normally H using the Cation entry Any terminal groups defined in the Compound Database will appear in the list of terminal groups see Creating a compound database on page 569 Calculations involving cyclic peptides For a hypothetical linear peptide KRATOSCIRCLE digestion with an enzyme which cleaves on the c side of arginine R as shown assuming no misseq cleavages n KRFATOSCIRICLE c will result in the three fragments KR ATOSCIR and CLE The sequence calculator has the ability to perform calculations based on theoretical enzyme digestion of cyclic peptides To
325. ion mass window Figure 27 10 Still holding down the mouse key position the reference peak in the centre of the display and release the mouse button see Panning using two displays on page 395 7 Click the mouse ADJ UST button on the centre of the peak in the display which matches the reference peak used in the calibration this will apply tolerance cursors around the peak Figure 27 10 Int 100 415 mV sum 41916 m Profiles 0 100 Smooth Av 10 Baseline 40 gas Apply tolerance cursor to the calibration peak 100 Expand the mass range around a calibrant peak 80 60 1889 74 1170 60 1342 87 1615 98 1690 96 400 600 800 1000 1200 1400 1600 1800 Mass Charge Figure 27 10 Setting up the second display for calibration Calibration of the instrument 471 472 Chapter 27 Instrument Calibration 8 On the Calibration window select the first reference peak in the scrolling list of reference peaks Figure 27 11 The selected reference will be highlighted E Calibration Select each reference peak in turn using the left mouse button Calibrant references E 000 019 Mass Angiotensin 2 Angiotensin 1 Observed mass at which reference TERRE OS peak occurs in the collected data 2465 20 ACTH_18 39 3657 93 ACTH_7 38 Press this button to get the mass at the current cursor position observed mass of reference peak Cursor mass
326. ion between the cursors x 10 Inserts the processing parameters between two cursors Shift Shift Ctrl Ctrl Amplify x 50 Amplify x Amplify x 500 5000 Amplify x 100 Amplify x Amplify x 1000 10000 Table 20 3 Display toolbar functions for text displays Normal action Copy the contents of the selected display into another Scroll columns to the right Scroll columns to the left Show the previous section of a multi section report Show the next section of a multi section report Zoom the selected display to the full window width Zoom the selected display to the full window height Zoom the selected display to the full window size Unzoom return a zoomed display to normal size The Display Toolbar Shift Ctrl Make an inset of the selected display between the cursors Show rightmost columns Show leftmost columns Zoom to 1 1 5 x Reduce to 2 3 current width current width Zoom to 1 1 x Reduce to 2 3 current height current height Zoom to show the next display in the series Chapter 20 Managing Data Displays Table 20 3 Display toolbar functions for text displays Continued Normal action Decrease font size by increasing number of lines in the display by 1 Increase font size by reducing number of lines in the display by 1 Shift Decrease font size by increasing number of lines in the display by 2 Increase font size by reducing number of lines in the displa
327. ions options Sample plates and plt files 123 Chapter 13 Preparation for data collection Alternatively the Select Well popup window available from the button to the right of the Well index selector allows the first last or a specific well to be dialled up See Figure 13 7 below Figure 13 7 Select Well window 124 Sample plates and plt files Chapter 13 Preparation for data collection 60600006000 Well Al Into Axima 64990000000 00000000000 00000000000 00000000000 00000000000 00000000000 00000000000 00000000000 00000000000 00000000000 00000000000 00000000000 00000000000 00000000000 00000000000 Left hand edge 00000000000 00000000000 00000000000 00000000000 0000000000000000 00000000000000008 3283888858888388 Well A24 Plate origin Bottom edge Figure 13 8 Indexing a standard 386 well sample plate For a plate with regularly distributed wells the Auto define wells button allows a block description to be applied for circular square or rectangular shaped wells See Figure 13 9 below Block sample well definition Wells across Wells down fi a Wells geometry Circular wells x Block prefix Definition of top right well in block Centre X mm Centre Y mm Circular well diameter mm Well gap mm Figure 13 9 Block sample well definition for regularly spaced wells Sample plates and plt files 126 Chapter 13 Preparation
328. ions to remain in the ion trap for manipulation However the ion gate calibration does vary with temperature of theAxima typically at 1000Da the drift is 0 1Da per 1 C Therefore if your experiment is using a wide gate the drift is not significant However if you are using the High 500 or Extra high 1000 gates the drift may be significant Button Low 100 Low 300 Mid 850 High 2000 Hi 3000 Resolution Wide 70 Std 250 High 500 Extra high 1000 Introduction Approx peak mass 200 Da 600 Da 1 700 Da 4 000 Da 5 000 Da 500 Da 7 Da 2 Da 1 Da Y Da Mass limits of the Mass Range buttons Typical range 100 to 400 Da 250 to 1200 Da 800 to 3 500 Da 1 500 to 8 000 Da 3 000 to 15 000 Da Approximate widths of the Ion gate Precursor ion mass 1 000 Da 2 000 Da 14 Da 28 Da 4 Da 8 Da 2 Da 4 Da 1 Da 2 Da Chapter 29 lon gate calibration Checking the ion gate The procedure below checks the ion gate at 1000Da using the Mid 850 mass range button Adapt to suit your requirements The plate carrier in the Axima has a well containing fullerite service engineers use this to check your instrument during preventative maintenance You can use this to check the ion gate calibration 1 Check the calibration of the Axima and if required redo the calibration 2 Open the QIT ToF MS window Acquisition gt QIT ToF MS click the Clear All button switches off the ion gate and then cl
329. is appended to the current data If you wish to edit the data see Creating editing a list of masses on page 324 322 Importing a list of masses Chapter 19 lon finder Pasting data from the clipboard 1 Set the lon Finder settings window to interpret the data that you are going to import 2 In the application select and copy the data In the example below the data resides with Microsoft Notepad P mass list txt Notepad Mi Ea File Edit Format View Help jolt Finder BBE i i Add entry Import data X Remove entries Remove all Results file destination Eolder Folder file Filename I Auto select filename Dialog data Load new Save Process Figure 19 6 lon Finder window showing data If mass tolerance data already exists in the lon Finder window new data is appended to the current data If you wish to edit the data see Creating editing a list of masses on page 324 Importing a list of masses 323 Chapter 19 lon finder Creating editing a list of masses The lon Finder window allows you to create new entries and edit current entries You can also remove entries Creating an entry New data appends the current data 1 In the lon Finder window select the Add entry button the mass cell is activated 2 Type in the new mass jolt Finder x Ion search template se Paste data Import data Remove entries Rem
330. is loaded next time press Save If the colours have been modified for a temporary change and the previous colour scheme is required to be reinstated press Load and the previously saved scheme will be loaded Press Load defaults to use the factory defined MALDI MS colour scheme Changing distribution trace colours Chapter 24 Choosing user defined colour schemes Changing common colours The Common Colour Editor window Figure 24 7 allows the user to define items which are common to all graph types These are namely the graph Axes colours the Header text colour the graph Labels colour and Peak markers colours colour Editor Common Axes colour gt Header text colour O O O Labels colour Peak markers colour O Colour sets Load Load defaults Apply Figure 24 7 Colour Editor window for Common items Changing these colours affects all graphical displays Having made all of the required selections press the Apply button to apply the selections to all of the displays This will apply the new colour scheme to the displays but the colour scheme modifications will not be saved in any way they are only temporary To save the user defined colour scheme so that it will be made the default colour scheme and loaded automatically when the MALDI MS software is loaded next time press Save If the colours have been modified for a temporary change and the previous colour scheme is required to be reinstated press Loa
331. is the identification of a protein assuming that it already exists within the database MS MS to analyse a peptide The desired result is the identification and sequence of amino acids in the peptide The database may also indicate where that peptide originated from Chapter 18 Protein peptide analysis using Mascot search engine Accessing the internet search form 1 If required log on to the Internet or your local Mascot server 2 From the MALDI MS window toolbar click the Internet Search lal x aje 2 3 x The Internet search window is displayed Internet search xi Internet search amp Select the engine to be used for your search from the list below Search engine Mascot PMF 7 Search settings This section allows you to specify a webpage URL to be used when submitting searches Once selected you can choose the type of database you wish to use Search URL http www matrixscience com cgi nph mascot exe 1 Database NCBInr bd Reporting Use these options to configure the sections of the report which the server creates for you You may also personalise your report with your name and e mail address Name Your name oo E mail address your name email_address co uk Report title Arabidopsis thaliana MS experiment E Search Cancel Accessing the internet search form 295 Chapter 18 Protein peptide analysis using Mascot search engine Mascot PMF searches
332. issed cleavages field select the tolerance From the Fixed modifications and Variable modifications fields if required select the appropriate modification s You can select more than one modification For the Protein mass field generally do not use this field as it may hinder the search For Treat masses as click the appropriate radio button In the Peptide tolerance field enter the required tolerance and in the Peptide tolerance unit field select either Da or mmu millimass unit from the drop down list In the Mass Type field select the required type from the drop down list For an automatic decoy database search select the Decoy checkbox Mascot MS MS searches Chapter 18 Protein peptide analysis using Mascot search engine MS MS specific criteria MS MS specific criteria hg Enter specific details to enhance the Mascot search e MS MS tolerance fos MS MS tolerance unit a H Instrument MALDI TOF TOF Peptide charge 1 v I Use ICAT quantification In the MS MS tolerance field set the fragment tolerance and in the MS MS tolerance unit field select either Da or mmu millimass unit from the drop down list In the Instrument type field select from the drop down menu e Axima Performance select MALDI TOF TOF e Axima Confidence select MALDI PSD In the Peptide charge field select the required precursor charge from the drop down field Only tick the Use ICAT quantification tick box
333. ist of calibrant references is complete type in a name for the calibration reference file and press the Save button The reference file will be written to the reference file folder A reference file can exist with the same name for both positive and negative ionisation modes concurrently e g insulin for both positive and negative ionisation Since the system Creating a new reference file Chapter 27 Instrument Calibration automatically generates file names based on both the reference name and the current instrument mode see Table 27 1 on page 463 this practice is acceptable Creating a new reference file 465 466 Chapter 27 Instrument Calibration Loading a reference file Having created a number of calibrant references these can be edited at a later date as above with new reference masses being added or unwanted references removed Simply remember to save the file after it has been edited To load a reference file press the List button on the Reference Editor window Figure 27 3 A scrolling list of available reference files will appear Figure 27 5 Reference files 1 1x Look in e References x ex Ea Fe 4 protein calstd pos_ref E angio2_fragments pos_ref My Recent E BSA pos_ref Documents E Cytochrome_C pos_ref CG E Glufib_cyano pos_ref fi LC MALDI Mix pos_ref Desktop E P14R_cyano pos_ref E peg pos_ref E peg_internal pos_ref r peg_na pos_ref peg li pos_ref pe li pos_re
334. it may be prudent to remove any unwanted data or compress previously collected data which resides on the hard disc Monitoring data collection Once the laser starts firing data will be displayed in the base window The frequency with which the display is updated depends on the settings made on the Display options window available from the MALDI MS View options Using this window it is also possible to request automatic printouts of data during data collection Most instrument controls e g power and aim can be modified during data collection Controls which cannot be changed become grey and cannot be selected Should it be necessary to suspend Firing the laser eoocecene al 152 Chapter 14 Collecting data from a sample Firing the laser or even abort a run during data collection the Suspend and Abort buttons can be used When data is not being stored during collection the data collection buffer can be reset at any time by pressing the Clear data button The profile count will be reset to zero After data collection When data collection is completed if Door control on the Experimental Technique window is set to Automatic then the sample stage will be presented for the next slide to be inserted Otherwise the instrument will await manual control If data is not being written to disc the Store button can be used to allow any data collected to be saved as a new run The Data files window will be displayed as abov
335. itor Compounds Name tof Saye ance Cursor mass Time Load named calibration 7 Load m Fragment fit Parent mass fo oooo ae setup Calibration I Auto calibrate T Fit through zero Correct Load Save Introduction Figure 27 1 Calibration window Chapter 27 Instrument Calibration Calibrant reference files A calibrant reference file contains a list of reference masses which can be used to calibrate the instrument For best results the reference masses should span the mass range in which the instrument is to be used For example to obtain an accurate molecular weight of a peptide fragment of mass 2846 Da Melittin the reference file used to calibrate the instrument should have peaks which occur at masses in this region both above and below the expected analyte molecular weight The instrument is calibrated by fitting a line through all of the reference masses in the reference file the closer the reference masses are to one another the better will be the predicted mass at any point between them Fragment ion of interest Figure 27 2 Calibrant references bracketing the required mass The calibrant reference file can optionally contain a comment or molecular formula for each entry A comment can be up to 80 characters of alphanumeric text which is either the name or a description of the calibrant reference e g Bovine Insulin Introduction 461 Chapter 27 In
336. ives an indication of the quality of the calibration and the reliability of the results given using the calibration Calibration of the instrument 477 Chapter 27 Instrument Calibration least squares fit through references Time reference mass Am a predicted mass yo Mass Figure 27 18 Calculation of the deviation from least squares fit For each reference point the difference Am in Figure 27 18 is calculated and plotted in the calibration graph against mass This graph is obtained by setting Display on the base window to Calibration Figure 27 19 dM amu 3 1000 2000 3000 4000 5000 Mass Charge Figure 27 19 Calibration graph 478 Calibration of the instrument Chapter 27 Instrument Calibration The data set for which the calibration graph is to be plotted is selected on the Display contents window Figure 27 20 Two plot types are available Absolute and Relative An absolute plot is a graph of Am against m whereas a relative plot is Am m against m The units for the Am axis are automatically chosen to be Da milli daltons ppt or ppm depending on the magnitude of Am Calibration Contents BE Dataset Trace Sample 1 angio2_msmsOl 7 G dM units Absolute Figure 27 20 Display contents window for calibration graphs Calibration of the instrument 479 480 Chapter 27 Instrument Calibration Nonlinearity correction The Axima Assurance and Co
337. k on the item a second time Wi Shimadzu Biotech Launchpad E CI Calibration g C Comments MQ Data vC 04 March 05 vi Andy vi Beam blanking C Bill vC chromatography C Default C imaging C linear mess vy Martha er ner 00 4 luk marl00004 TF Figure 32 3 Items selected for archiving When viewing folders in the tree list a ticked box on a white background indicates that all the items within this folder have been selected A ticked box on a grey background indicates that only some items in this folder have been selected If the option Show file information is selected then with each new selection the files selected are counted and the file totals and file sizes appear in the bottom of the window Figure 32 4 File information File totals marl 00001 14 58 Mb in 296 files 546 bytes in 7 files Created 30 Jun 2005 Show file information IM Figure 32 4 File information shown on the Archiver window If this option is not selected this will soeed up the archive selection process Having made all of the required selections then the process of archiving to the archive medium can begin To clear all selections made and start again press the Clear button any selections made will be deselected Chapter 32 Archiving data Archiving to removable disk media It is best to have storage media pre formatted ready prior to archiving as the archiver is unable to format unformatted media Press the Archive butt
338. ker gt lt MinMass unsigned integer gt lt MaxMass unsigned integer gt lt Minlsotopes unsigned integer gt gt 0 lt MaxVariationPercent unsigned integer gt 0 100 lt Overlapping enumeration gt true false lt MinOverlapPercent unsigned integer gt 1 100 Block lt MSx gt Processing gt lt PeakFiltering gt lt FilterValue string gt Value can be formula or floating point value lt FilterTolerance float gt lt ToleranceUnits enumeration gt Da mDa ppt ppm lt FormulaeAre enumeration gt Average MostAbundant Monol sotopic ASCII Text Method file format Chapter 15 Automated operation Multiple FilterValue values are allowed Block lt MS1 gt lt Processing gt lt Mascot gt lt Title string gt lt Database string gt lt Taxonomy string gt lt Enzyme string gt lt MissedCleavages unsigned integer gt lt FixedModification string gt lt VariableModification string gt lt ProteinMass unsigned integer gt kDa lt PeptideTolerance float gt lt PeptideTolUnits enumeration gt Da mDa ppm percent lt MassValues enumeration gt MH M lt Overview enumeration gt true false lt ReportLength unsigned integer gt Number of entries to report lt MaxSearchPeaks unsigned integer gt Number of peaks to search Multiple FixedModification and VariableModification values are allowed Block lt MS2 gt lt Processing gt lt Mascot gt lt Ti
339. l technique tab 120 Introduction Chapter 13 Preparation for data collection Sample plates and plt files The Axima instruments use a set of standard 96 or 384 well micro titre plates A physical description of the layout of the wells ona sample plate is passed to acquisition via plate plt files The slide window allows the relevant plate file for a sample plate to be loaded and gives access to a plate file editor where plates files can be created or edited and so provides the means to customise the physical dimensions and positions of wells on a sample plate for a particular experiment Acquisition Firing Exp Tech Auto Quality Storage Slide Raster Tuning Sample plate file Change Plate Carrier Edit sample plate Align plate Load Slide loading Manual hd Slides in series po g Next slide after 100 seconds Apply Initialise Stage Figure 13 4 Axima Slide tab on the Acquisition window Sample plates and plt files 121 Chapter 13 Preparation for data collection Close up showing Ee CWIXY UY 0 Ul UOIDIJIG 1 3 4 5 678mg 1010101010100 010 OQOQO0Q0000 OFC OOQOQO0Q0C0COC OFC amp OOOOOO0O0OOO L wWiselololererererere TT OLOLO1O101 0101010 GOOOQOOQO0Q000O0OO TO LOL lOl 1O1O10 0 1 OOOQO0000O0C wells Al to D4 0 2 J 2 J J J 2 Figure 13 5 A 384 well titre plate Sample plates and plt files
340. labelling 101 0 Figure 22 1 Accessing the Peaks toolbar 3 Select the Peaks toolbar option the toolbar is displayed aly gt lt px Peak labelling 409 410 Chapter 22 Manual peak labelling Table 22 1 Peak toolbar icons Function Pin toolbar if you intend to label delete several peaks select this icon to keep this toolbar available otherwise the toolbar will disappear each time you use it ae Remove toolbar this icon appears when you select the above Pin toolbar icon Select this icon to remove the toolbar Insert peak label labels a peak between two cursors Delete peak label deletes a peak Delete peak labels deletes a range of peaks between two cursors KKK B Inserting a peak Peak labelling 1 Position cursors click mouse middle button either side of the required peak Position cursors lnt 934 EN 64900 mV Profiles 1 59 Smooth Gauss 10 Baseline 30 14 ojx 102 4 101 0 101 5 102 0 102 5 103 0 Mass Charge 2 Select the icon the peak is labelled the label may not appear on the spectrum depending on the Peak processing parameters and Display option you have set but it will appear in the mass list Chapter 22 Manual peak labelling Also when you select this icon the Manual peak assignment feature becomes available to you in the Windows toolbar usually at the bottom of your screen 37 Manual Peak Assignment 3
341. late Cation Proton H m Digest Length 15 Sequence Calculator olx File Edit View Settings Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Arg Long symbol notation 15 10 I 4 gt gt gt I Synchronise Name fUntited Masse Resolution f oes TEER g N terminus Hydrogen H 7 Monoisotopic 7 24 C terminus Hydroxy HO x Fragmentation Singly charged z Generate Peak Markers Calculate Cation l Proton H x Digest Length 15 Figure 42 7 Example of sequence notation in the view window Select the desired sequence notation to be displayed 608 Introduction Chapter 42 Sequence Calculator Including linked sequences The sequence calculator has the ability to calculate the molecular weights and fragments of cross linked chains where one sequence is linked to another either through Cysteine cross links or by any other means The calculations can be made to exclude or include linked chains This will be discussed further in section on page 616 Reporting decimal places The Sequence Calculator has the ability to produce a text report based on the current sequence or loaded sequence from a database see Sequence reports on page 620 The calculations will report up to 9 decimals depending on the option selected in this field Importing sequences from ASCII files To import a sequence from an ASCII text file the file must be i
342. lay Chromatogram Profiles 1 2000 Mg Masses 1 7504 l gt I MM 7 61 r Data mar100004 10 Mar 1993 19 06 Factory calibrated PSD of 1046 51 Shimadzu Biotech Kompact MALDI 3 2 7 0 Build 20050509 Linear High Power 45 100 0 0 mY mass 1 to 7504 1000 1200 1400 1600 1800 2000 Profile Profile 873 Aim 450 Power 45 Profiles 774 to 873 Aim 400 to 450 Power 45to45 NUM Figure 30 6 Using cursors ee a chromatogram pea Having set the cursor positions to the start and end of a peak on the wae clear the peak list by pressing Clear and press the T4 cursors button on the Chromatography window The peak delimited by the cursors will appear in the detected peaks list Repeat this procedure until all of the required peaks have been marked Figure 30 7 510 Chromatographic peak detection Chapter 30 Chromatography AF Chromatography Oxi Mark peak start and end Detection HH Dataset 7 mar100004 with cursors on the Mass range 300012000 ah t chromatogram display and Method Cus z Td press this button to record j Signal the peak position Smoothing Average x Average rE Segments 20 P Combine Peak width 4 Profiles Peak height 1 4 my Detect peaks Peaks detected pde F E Ascending Profiles E Both Poea Manually entered peaks will Clear tags appear in the peaks detected list soe os FR Ezi Apex n
343. le Retry loading the file the file may have been corrupted or may contain invalid information Unable to open file Check that the file exists in the selected directory There was an error reading the parameters file Defaults have been assumed Retry loading the file the file may have been corrupted or may contain invalid information If the error persists you will have to open another parameter file and delete the file giving the error The default values will allow the program to operate normally This is normal if the default parameter file tof parameters has been deleted An error occurred while writing the parameter set Retry the operation check file and directory write permissions on the directory being written to and check disk space The requested dataset does not exist Check that the dataset exists in the selected data directory and that all files in the set exist run cal raw stats The requested dataset has an invalid filename Check that the filename has the format the name 4 digits extension run i e MYNAMEOOOL run Maximum datasets loaded Please unload an existing dataset first 10 datasets have already been loaded unload a dataset in order to load a new one Cannot gain access to the data directory Check the network or hard disk integrity is the data directory a valid directory An error occurred whilst storing data Retry the operation check file and directory write permissions on the
344. le plate Algnplate Load Manua Ade loading Sides in series Next siie alter 100 CTET Fring Exp Tech Auto Quoty Storage Side Raster Tunng Load Save Reiter Type Ragin Rectwwde F Faster Style Ty Raster Z Wid fs am Height 116 00 um Cente 00 um CeweY 00 un Fors 120 Calculate Raster Based n Spacing Spacing i000 un Figure 5 9 Acquisition menu tab options a Instrument menu Chapter 5 Window and menu guides Acquisition Reflectron Firing Bip Tech Auto Qualty Storage side Raster Tunng me EET a seve Votages PE settings Saro P i wegen TA sotto TO wes F swrn E hon TT oe Bisedat A ane Bort Erd er oe epee I e eE Linear detector Refl y defl 4 E pen geteer A on i e Sn ane J ane Figure 5 10 Acquisition menu tab options b The figures above shows all the tabs available for the Axima Performance instrument the number and contents of the tabs vary for other instruments and are more fully described in the sections on preparing for and collecting data Instrument menu 45 Chapter 5 Window and menu guides Automation menu This menu provides access to the different methods available for automating data collection and analysis For details of the Method editor and Auto experiment see Introduction on page 166 i Method Editor default mtd Sample Me
345. le that the correction function may make matters worse In this case monitoring the mass errors in the calibration plot window is recommended see Figure 27 19 Nonlinearity correction Chapter 28 Fragment ion calibra Chapter 28 Fragment ion calibration tion 482 Chapter 28 Fragment ion calibration Introduction Introduction You should not normally need to perform fragment ion calibration Once the fragment ion calibration has been set at the factory or by a service engineer the calibration is usually stable Only attempt this procedure if instructed to by Kratos or Shimadzu service centre Fragment calibration is only applicable to Axima Performance and Confidence models only The Axima Performance is fitted with a CID cell which introduces a collision gas helium in to the flight tube This gas aids fragmentation The Axima Confidence relys on the time of flight through the flight tube to allow fragmentation Therefore the fragmentation within the Axima Performance is more pronounced To perform MS MS monitoring experiments using the ion gate the instrument must have another calibration called the fragment ion calibration This allows the instrument to assign masses to the daughter fragments which have arisen from the decomposition of the same parent ion The fragment calibration is a function of the mass ratio of the fragment ion to the parent ion Hence the one fragment calibration is correct for all values
346. le traces can be shown with separate X axes under each graph or with a single X axis on the bottom graph as in Figure 20 30 Customising Graphical Reports Chapter 20 Managing Data Displays j 400 i 600 Separate X axes 109 96 110 24 70 06 255 22 72 10 343 10 506 22 619 27 4 49 aci 28 400 09 5h 200 400 600 Mass Charge Single X axis 255 22 343 10 506 22 619 27 264 28 400 09 591 24 200 400 600 Mass Charge Figure 20 30 Graphs axes options Graph Headings Figure 20 32 shows a graphical display with all of the heading and other graph options enabled in contrast the graphs in Figure 20 30 have the headings disabled The graph heading can be switched on and off by ticking the Heading box on the Graph Customising Graphical Reports 373 Chapter 20 Managing Data Displays Options property page Ticking the relevant boxes enables the individual options in the graph heading Figure 20 31 shows the names of the component parts of the graph heading Display title Display 1st comment Display data and cal Data bill angio2_msms0006 G c 4 Mar 2005 14 37 Cal tof 4 Mar 2005 14 37 CID of 1046 51 Biotech Axima ToF V2 7 Mode reflectron_ms_ms Power 73 Gate 1035 00 1060 00 P Ext 1060 bin 53 12 m sum 627 mV Profiles 1 54 Smooth Av 5 Baseline 80 Display folder ee heading Figure 20 31 Components of the graph headings Display instrument Title line text Prefix text
347. lect Peaks Manual Peak Assignment Internet Search Figure 30 1 Processing menu Chromatography option The Chromatography window will be displayed Figure 30 2 Chromatographic peak detection can only be used where data has been stored for individual profiles or averaged profiles Introduction Chapter 30 Chromatography Chromatographic peak detection Having collected data select the desired mass range within which to search for peaks using the Mass range entry Three different methods of peak detection can be used Threshold Gradient and Cursor The Threshold and Gradient methods need little description as these are the same methods employed in processing on the Peak clean up window Cleaning up data on page 235 and have already been discussed The Cursors method is a manual method for specifying peak positions and will be discussed after automatic peak detection The Signal option specifies whether peak detection is based on the Largest or Average signal in the profiles The same range of smoothing options apply to chromatographic peak detection as to the peak clean up options For an explanation of how smoothing of the collected data is carried out see Cleaning up data on page 235 Chromatographic peak detection 506 Chapter 30 Chromatography The options available are Average Gaussian or Savitsky Golay smoothing AF Chromatography x Detection BBB Dataset 1 pep_mix 246640005
348. lect Peaks button as shown in Figure 15 20 below Method Editor 185 186 Chapter 15 Automated operation Method Editor Compare Sequence Options Search Currently loaded sequence Select Peaks Match Settings Missed theoretical peaks V Missed experimental peaks Tolerance 2 Da Si Sequence Select Peaks x Font Medium f Short symbol th Peaks 25 4 a YGGFLRRIF Cancel 5 Figure 15 20 Compare Sequence Select Peaks window Type the mass range in the Masses entry over which peak selection is to be performed The number of peaks to select is specified by the Peaks option Cursors can be used to select a range of masses from a spectrum display Position two cursors bracketing the mass range of interest and press the fJ button Masses will be set to the range marked by the cursors The most intense peaks in the mass range will be chosen When the currently loaded sequence or a single selection in the Load Sequence window has been made the Missed Theoretical peaks and Missed Experimental peaks options can be ticked Select the missed theoretical peaks box to display the missed peaks that where expected to appear in the sequence match report Select the missed experimental peaks to display the actual peaks that where missed during the sequence report Select a Tolerance to be used in the peak matching peaks which are outside this tolerance window will not be matched To search through a data
349. les 1 142 Sthooth Av 20 Bag lnt 1 5 mY sum 220 mW Profiles 1 142 Smooth Av 1297 16 1297 16 1298 1300 134 1300 Mass Charge Mass Charge For Help press F1 Z Figure 20 9 Resizing using the splitter bar cursor Multiple displays Wint 100 Chapter 20 Managing Data Displays Scrolling graphical displays Each graphical display can contain a single graph or a number of graphs These graphs can be scrolled panned marked and annotated as required There are a number of display toolbar buttons which facilitate scrolling and selection of the graph s mass range These buttons are shown in Table 20 2 on page 338 The two scroll graph buttons and are used to scroll the graph data across the display by 10 in the direction of the button arrow Left is towards lower values and right towards higher values The button shows the full range of data i e on a spectrum it will dispiay the whole mass range Figure 20 10 The operation of the Tt display next S display previous and EJ display all data buttons depends upon the type of display If the display is showing a mass range for a profile of collected data then and would show the data for the next and previous shot respectively In this case the button would average the data for all of the shots and show this average On a chromatogram display showing the signal intensity for a given mass the T and buttons would show the
350. lications oe Compare Se Modification oer Delete Min Loss Primer ja i n 9 Analud insert vv 1g hh eo Las fiso Clear Max Gain Primer x p O Print Tolerance Lower limit Upper limit M bad E t gt ow he E Pa z Peak areas of Primer 15 2 Primer modifications table i lon Finder me cima Noise from Peak S N ratio AE I Medien of non peak signal 7 a JE Acquisition Peak resolution 50 H 70 i BE Peak Select 5 E EH Raster 5 Epe M EN 1 3 Laser Firing Spectrum j Print aeaa O MS MS 2g Processing META files Spectra Lowless 20 detected gs Peak Cleant Overwrite High plus J OPE Monoisotopi existing files E 20 a detected CIEE Peak Filterin Results a 192 Mascot text file C Program Files Shimadzu Biotech Launchpad Export daoligo billoligo txt_ Browse Wy Output M gt Data Storage Get enue setma Save Method Load Method Figure 15 22 Method Editor Oligo Analysis The various other parameters required are entered in the Oligo Analysis window of the Method Editor as shown in Figure 15 22 The modification masses and their corresponding maximum allowable percentage areas of the primer mass peak are entered in the Losses list box N B The modification peaks actually detected and reported are those at the primer mass plus or minus the masses entered in the list box To enter a modification in the list box enter the mass and percentag
351. lick the f J cursors button on Chapter 25 Automatic graph labelling scaling and printing the Display Options Peak Labels property page The nearest peak to the cursor position will be labelled with its mass all other peaks will be labelled relative to this peak Figure 25 9 Select peak with cursor Yolnt 100 6 m sum 637 mV Profiles 0 100 Av 10 Baseline 40 86 94 5550 5600 5650 5700 Mass Charge Figure 25 9 Relative peak labelling Labelling peaks 453 Chapter 25 Automatic graph labelling scaling and printing 454 Labelling peaks Chapter 26 Getting a Summary of Run Conditions Chapter 26 Getting a Summary of Run Conditions 455 456 Chapter 26 Getting a Summary of Run Conditions Summary of run wide conditions Data can be collected from sample slides under quite different operating conditions A summary of the conditions and settings used when data was collected can be obtained by setting Display to Summary Select Display contents from the View menu Or click on the toolbar display contents button The Summary Contents window will be shown Figure 26 1 Eq Summary Contents Bik Datasets 1 angio2_ mems00q _2 p14 msms0004 4r_msms0004 None aw Figure 26 1 Summary Contents window Select the datasets for which summary information is required press All to select all datasets or None to clear selections Press Apply after making sel
352. licking the mouse SELECT button within the inset and modifying display parameters in the same way as for any other display It is also possible to transfer data into or out of the inset by using the copy display feature described in Copy insert and delete displays on page 365 The Display Toolbar buttons also operate on data within an inset with the exception of the zoom buttons which act upon the insets parent display e g pressing the full size zoom whilst an inset is selected makes the parent of the inset full size Multiple insets can be created within a display and insets may even be created within other insets The size and position of an inset may be changed after creating the inset either by using the splitter bars as outlined in Resizing displays on page 347 Copy insert and delete displays 369 Chapter 20 Managing Data Displays Deleting inset displays Select the inset display to be deleted From the Display menu select Delete and from the Delete menu select Inset and the selected inset display will be deleted Figure 20 27 Paste Insert gt Colum Row Annotate ee Tags gt All Insets Peak labelling gt Figure 20 27 Deleting an Inset Display Copy insert and delete displays Chapter 20 Managing Data Displays Customising Graphical Reports The Display Options window allows each individual display to be customised to show only those features which are required Click on Options
353. ll appear Figure 41 2 598 Chapter 41 Defining Enzymes Enzyme Database BEI Help Enzymes AspN Chymotrypsin LysC Trypsin Enzyme Chymotrypsin ose Cleavages C side of F not adjacent P C side of Y not adjacent P C side of W not adjacent P C side of M side of L CSide oF o Not Adjacentto P Insert Delete ok Cancel Figure 41 2 Enzyme Database window To create a new entry in the list type the name of the enzyme after Enzyme and press Insert To remove an entry from the list select the entry with the mouse SELECT button and press Delete The enzyme cleavage rules are defined in the lower part of the window To define the rules for enzyme cleavage an enzyme must be selected in the top half of the window Select either the N side or the C Side this defines which side of the peptide chain the enzyme will cleave this is either the N terminus side N side or the C terminus side C Side The amino acid at which cleavage takes place is specified as the N side or C Side Of another amino acid Enter the amino acid at which cleavage occurs Rules governing cleavage when certain combination of amino acids are present adjacent to the cleavage site are specified using the Adjacent option There are two possible conditions cleavage will Not take place when any one of a set of amino acids is adjacent to the cleavage site or 600 Chapter 41 Defining Enzymes e cleavage will Only take
354. ll service Press OK to reset instrument Error 23180 There is a problem with the ion trap gauge Applicable to the Axima QIT model only Please call service Press OK to continue pumping Error 23190 There is a problem closing V5 after another error Applicable to the Axima QIT model only Please call service Press OK to reset instrument Error 23200 The room temperature is too high Room temperature is too high or the airflow fans have failed Try cooling the room and press OK or call service Error 23210 The room temperature is too high Room temperature is too high or the airflow fans have failed The vacuum system in the instrument has been shut down Try cooling the room and press OK or call service Error 23220 The temperature of the RF electronics is too high The instrument has been shut down Please call service Press OK to reset instrument Error 23230 Distribution board communications error Please call service Press OK to reset instrument Error 23240 There is a problem with the laser Laser Type nnnnnn Error Code xxxxxxxx Please call service Press OK to continue Error 23250 There is a problem with the instrument comms If this problem persists please call service Press OK to continue Error 23300 Instrument not in a valid vacuum state This error implies that there is an instrument fault The valves and pumps are not in any known state Switching the Axima off and then on using either a switch at the mains supply or t
355. lows a database to be searched for matching keywords e g Cytochrome or Chimpanzee matching amino acid sequence e g ATAQQ and e allows the molecular weight of the sequences being searched to be restricted e g only search through sequences in the range 240 1033 Da Firstly select a database by pressing the Select button The window shown in Figure 42 10 will appear and from the list select the database to be used Set Files of type to be the desired database format Select Sequence Database BEI Look in jo Peptides x ef fe SyactH 1 17 ksq ACTH ksq My Recent ei angiotensin 1 ksq ESTEE eH angiotensin 2 ksq A Z angiotensinogen ksq Desktop E default ksq ra E pi4r ksq E standards ksq My Documents My Computer T a Cri File name X Places Files of type l Kratos format ksq X Cancel Introduction 6ll 612 Chapter 42 Sequence Calculator Introduction Figure 42 10 Peptide databases list In the Load Sequence window enter the Keywords Sequence and Mass range as required Any number of keywords can be used the search is not case sensitive and will find a keyword contained within a larger string e g searching for ORP will find the keyword in Dynorphin Where the field is left blank all entries in the database will be displayed This technique of restricting the search can be beneficial in that the more precise the search restrictions are the better t
356. lso display relevant information about how you might avoid seeing the error again The complete list of fatal errors with corresponding descriptions and possible corrective actions are described in the following tables The first table describes the error numbering structure and the second table details all errors Prefix Switches 1 None 2 mzdata or mzxml 3 ior o 4 p 5 yal 9 Any 0 Any Error Classification File specification errors Output format errors Input and output directory errors Parameter file errors Log file errors Serious operating system failures Command line errors Table 34 6 Error numbering structure 101 No run files matching your specification were found 102 Not a valid file specification 201 No output format specified No files matching the specification could be found in the path given An input file specification contains invalid characters The path for individual input files should not be specified since it is incompatible with the i and s switches Instead one input path should be specified using the i switch You must specify either mzxml or any derivatives or mzdata Table 34 7 Errors Using the command line editor 202 203 301 302 303 304 305 306 401 402 403 Invalid XML contents specified Too many output formats specified The input directory does not exist Too many input directories Too many output directorie
357. luding at least a Laser Firing node in the method tree The Oligo Analysis node must be selected Oligo Analysis requires that a sample identification code ID and a primer mass be supplied for each sample well analysed these are input via an ASCII experiment file The file consists of a single line for each sample well to be analysed The file can also optionally specify a data file a method file and a flag to suppress printing options for the current sample An oligo specific example of an ASCII experiment file might be as shown in Figure 15 21 Don t print spectra or Sample well Al other method print a a options for well A1 A1 Idal primer 3048 91 Mmoprint Use 602 14 Da as the primer mass for B1 Idbl Iprimer 602 14 me B1 A2 Data file for Well A2 Sample ID for well A2 Method file for well A2 Method Editor Figure 15 21 Part of an ASCII experiment file in Oligo Analysis Chapter 15 Automated operation A suitable ASCII experiment file may be generated from either PC DOS or UNIX based computers the general file format is as described in ASCII text experiment file formats on page 210 Method Editor oligo3 mtd lf x Sample Method I Sample Method oligo3 mtd Oligo Analysis a EY Parent El MY Acquisition a 333 Raster OvX Auto Quality Primer Mass Modification Max of primer 3 Laser Firing Processing OL Calibration EX Peak Cleant ME Monoisotopi CIEE Peak Filterin 182 Mascot MY App
358. ly skips sample spot numbers for which no data was collected There is the option of collecting positive negative ion data and neutral fragment spectra Both the charged ion spectra and neutral spectra can be displayed by clicking on j2 for charged spectra and or m for neutral spectra To remove any dataset traces from the current display simply de select the Trace option for the traces to be removed With the introduction of the Axima series of instruments one click changes of dataset or sample have been introduced to the Spectrum Display contents Thus the display is automatically updated without the need to select unselect or apply menu items Sometimes it is useful to be able to display more than one sample from a particular data set this is especially true for Axima Confidence data sets this is achieved using the Display multiple sample option and is described in Introduction to displaying data on page 107 Selecting the data for processing Many of the sub windows in the MALDI MS processing suite process the collected data e g calibration polymer analysis chromatography Each of these windows needs to know which dataset to process This is achieved using the Process option on the Spectrum contents window One dataset from those loaded can be selected for processing using the H and n process buttons Each of the sub windows which uses a dataset for processing will show the name and trace colour of the data
359. m Files shimadzu Biotech Launchpad pata arb o5 apr 06 tof2_mix_refoo10 Units Mass Tolerance Da Intensity Counts Search mass 757 Tolerance 2 Nearest mass to search mass in range 757 Intensity 3328 Largest intensity mass in range 758 Intensity 7680 All masses Mass Intensity 757 3328 758 7680 Search mass 1046 Tolerance 1 Nearest mass to search mass in range 1046 Intensity 1024 Largest intensity mass in range 1046 Intensity 1024 Al masses Mass Intensity 1046 1024 Search mass 1297 Tolerance 1 Nearest mass to search mass in range 1297 Intensity 9984 Largest intensity mass in range 1297 Intensity 9984 All masses Mass Intensity 1297 9984 Search mass 1535 Tolerance 2 Nearest mass to search mass in range 1534 Intensity 11008 Largest intensity mass in range 1534 Intensity 11008 All masses This example is highlighted to differentiate the areas of interest Processing the data 331 332 Chapter 19 lon finder Header information The file contains the name and path of this file followed by the path and name of the spectrum run file The units for the results are defined on the next line Results information Results for each target mass include Search mass Tolerance Nearest mass and intensity to the target mass Largest mass and intensity within the tolerance range List of all masses and their intensity within the tolerance range Processing the data Chapter 20 Mana
360. ma is operating Please call service Press OK to continue Error 23070 Could not close gate valve This error is only applicable when the Axima is powered up Please call service Press OK to enter vented state Error 23080 Could not rough source Please check door and or call service Press OK to continue Error 23090 Could not pump source Please check door and or all service Press OK to continue Error 23100 There is a problem with the backing gauge Please call service Press OK to continue pumping Error 23110 There is a problem with the backing pump Please call service Press OK to reset instrument Error 23120 The instrument is overheating The backing pump has been disabled Please call service Press OK to reset instrument Error 23130 Attempts to pump the sample chamber have failed Please check the door seal Error 23140 There is a problem with the variable capacitor Applicable to the Axima QIT model only Please call service Press OK to reset instrument Error 23150 There is a problem calibrating the laser power Applicable to the Axima QIT model only Please call service Press OK to reset instrument Error 23160 There is a problem calibrating the sample stage Applicable to the Axima QIT model only Please call service Press OK to reset instrument Error messages 657 658 Chapter 44 Summary of error messages Error 23170 There is a problem sending the stage to the load location Applicable to the Axima QIT model only Please ca
361. madzu Biotech Launchpad Programs config_environment exe 2 If you change the URL the Refresh button is displayed Click it to continue 3 At the Database field select the required search engine from the drop down list MSDB Comprehensive non identical protein database NCBI nr Comprehensive non identical protein database EST_human Human subset of GenBank EMBL DDB sequences from NCBI EST Division EST_mouse Human subset of GenBank EMBL DDB sequences from NCBI EST Division EST_others SwissProt a high quality curated protein database Random Random sequences for verifying scoring Mascot MS MS searches statistics Reporting Chapter 18 Protein peptide analysis using Mascot search engine Reporting Use these options to configure the sections of the report which the server creates for you You may also personalise your report with your name and e mail address Name i E mail address your name kratos co uk Report title Arabidopsis thaliana MS MS experiment mz 1722 Top hits o repgrt 20 d pfoduce n oyerview section SMATRIX SCIENCE User Email Arabidopsis thaliana MS MS expriment mz 1722 Overview Table Click on column header to jump to entry in results list Move mouse over any indicator to highlight identical peptides Click on an indicator to see details of individual match Use check boxes to select sub set of queries for new search Mouse ov
362. matically so that the largest signal in the data represents 100 intensity and all other peaks are shown relative to the largest peak This is achieved by setting the Profile scale to Automatic However when tuning the instrument or adjusting the laser power setting it is useful to be able to monitor the increase in signal level For this reason the various scaling options were provided To set the scale of the graphs manually set Profile scale to Manual and type in a value up to a maximum of 2500 mV for the required scale of the graphs The display will reflect decimal scales of lt 10 00mvV higher values are displayed rounded to the nearest integer yet the decimal value entered is still applied At the top of the graphs the graph title indicates the value to which the traces are scaled in the same colour as the trace itself and in the order in which the datasets were loaded Isometric plots Chromatogram style displays which rely on three dimensional isometric plots can be customised using the sometric plots options The angle of the isometric projection or viewing angle Customising Graphical Reports Chapter 20 Managing Data Displays can be altered from between 5 to 90 The proportion of the display taken up by the base height can be adjusted from 0 to 99 as shown in the example in Figure 20 34 Base height 20 Vitti ee LMM AA QL A Gi MELLEL LEME f f VME na aa
363. ment ion calibration Table 28 3 Apparent masses the same P14R MS MS spectrum without the fragment calibration applied Some of the fragment ions particularly w ions will not be seen unless the CID gas is enabled and is pure helium at the correct pressure 1 In the Exp Tech window set Reflectron mode e Mass range 0 8 000Da Laser rep rate 10Hz e CID enabled 2 In the Firing window set 10 shots per profile e 50 200 profiles Blanking 300Da e Pulsed Extraction 1534Da 3 Collect a normal ms spectrum from a P14R sample Use gausssian smoothing of 20 and baseline subtraction 80 4 Calibrate on the P14R parent monoisotopic peak at 1533 86Da and the Cyano2H peak at 379 09Da Save the calibration 5 With the ion gate set to 1520Da to 1550Da collect 10 ten P14R fragment spectra settings as above all with the parent mass within lt 0 1Da of the correct value of 1533 86Da Aim to achieve good quality fragment peaks in all of the fragment spectra 6 Load all ten P14R fragment calibration spectra into the Maldi_MS data sets 7 Set the processing to data set number 1 in the traces window 8 Inthe calibration window switch off the fragment calibration by selecting remove in the fragment calibration mass section mid right hand side of the window This will apply the normal calibration to the fragment spectrum the fragment calibration is switched on automatically after collecting a spectrum wit
364. ment masses and vice versa The CID gas was not switched on so the wrong fragments were used The sample is not P14R residuals are more than 0 2Da then possible reasons are the wrong apparent mass has been assigned to that fragment the peaks for that fragment in more than one calibration spectrum are systematically high or low e g the wrong fragment isotope was found the reflectron is faulty HV breakdown or a faulty resistor calibration is acceptable then store the calibration to tof and close the fragment calibration setup Axima Performance 487 488 Chapter 28 Fragment ion calibration Examples of peak shapes The examples below represent the various different shapes of peaks that you can expect from ms ms fragmentation 1534 03 1339 75 100 100 f 1535 80 344 07 90 90 J 20 U V oo 70 70 ate 1436 71 60 so 50 1 aR 39 40 40 30 20 20 20 s 1699 36 1549 S 0 y A 1531 07 s E 5 10 AL AZ ee ee 0 2 DA 1530 1532 1534 1536 1538 1540 1220 1225 1340 1345 1250 MasyCharge Masscnarge 1145 65 951 54 100 100 90 L a7 bad 70 70 7 60 50 40 40 j 20 30 20 20 N 10 94468 oj eee a Soy oj gt E N en Ae 1135 1140 1945 1150 1155 oa 015 20 255 MasgChamge MassCharge 46 03 195 11 100 100 555 33 90 90 80 80 70 563 14 70 60 60 50 50 f 40 40 20 30 20 20 ii 10 541 56 n j N i 10 N A j TER A o V VW AS Was of
365. mentation depending on the sample The following example will gate the precursor so that only this ion enters the flight tube The ion then collides with the CID gas to produce the fragmentation ions Internet search Internet search e Select the engine to be used for your search from the list below Search engine Mascot MS MS hd 1 In the Search engine field select Mascot MS MS from the drop down menu Search settings Search settings This section allows you to specify a webpage URL to be used when submitting searches Once selected you can choose the type of database you wish to use Search URL se http www matrixscience com cgi nph mascot exe 1 Icon is displayed while MALDI MS attempts to connect to the server 1 The Search URL field is usually displayed automatically The Matrix Science database URL is http www matrixscience com cgi nph mascot exe 1 Mascot MS MS searches 303 304 Chapter 18 Protein peptide analysis using Mascot search engine If you are using a local Mascot server the URL is typically http lt mascot server name gt mascot cgi nph mascot exe 1 Internet or your Mascot server gt If you experience problems accessing either the Ask your IT department to check the access rights from the PC Check that the correct parameters have been set up in the Environment Configuration Editor default path to the editor is C Program Files Shi
366. mode Molecular formula C1012H1600N2620321514 Resolution 200 at 50 lnt 23301 36 JA 23100 23200 23300 23400 23500 Mass Charge Figure 23 4 Isotopic distribution of Trypsin Compensating for adducts When data is collected on an Axima instrument ions can be formed which have adducts attached The fragments produced and their ratios will depend on the preparation chemistry Theoretical spectra for peptides Chapter 23 Displaying simulated data Figure 23 5 shows the same formula as above with simulated adducts of H K Na and Cj 1H 0 a matrix compound Note that the trace for the molecule without any adducts is disabled as this is not an ion which will occur Distribution Contents BBE Formula Scale Molecule Vv Cii2Hie00N2820321S14 fico Adduct1 IV RO fo a Gain Loss Adduct 2 Iv Na jo a Gain Loss Aduts KE o o TP Gin Los Adducta MW fctHiof sd Gain Loss Simulated resolution jpo Auto mass range V Clear Molecular formula C1012H1600N2820321514 Resolution 200 at 50 lnt 100xM 30xM H 30xM Na 30xM K 20xM C11H1104 Sum 23312 05 100 23525 09 23100 23200 23300 23400 23500 23600 23700 Mass Charge Figure 23 5 Theoretical distribution of Trypsin with adducts When a number of adducts are simulated a separate trace is shown for each adduct in a different colour The sum of all of these adducts is also shown this is the peak shape which would be seen in data collecte
367. mple Test Mascot Search LC MALDI Accession Chain A Identification And Struct gi 6730143 96218 100 1880 000 86 Chain A Kinetic And Crystallograp gil62738464 97671 720 1880 000 86 cane of Grog ssn icnie E57 76060000 os For Help press F1 The main display shows you a graphical representation of the experiment results There are three levels to the tree structure Displaying Auto Experiment Results 223 224 Chapter 15 Automated operation e Sample Details about the sample like well ID and sample ID Tests The types of tests carried out on the sample Test Results The results of a test in a simple table format and what data files were used by the test Displaying Auto Experiment Results Chapter 15 Automated operation Using Auto Experiment Results viewer This section describes how to view the results interpret the results display the mass list display spectrum and Mascot results Viewing results e You can expand and compress tree nodes e adjust column widths Expand tree nodes You can expand any node that has a solid triangle in its bottom right hand corner AE0023_0001 318 1881_0001 MALDI MS of x File Edit View Instrument Automation Processing Help a bel Hae 2 8 x a4 Display auto Experiment Resuts x Profiles 1 16 Masses 1459 2426 KA C Program Files Shimadzu Biotech Launchpad Data Customers
368. ms ms spectrum in the apparent mass box c press insert to enter the peak into the list d if the peak was from the list the message box peak within 0 5Da will appear asking if you want it replaced press OK to accept the new apparent mass value 13 Save the fragment reference file e g as P14R_ peaks Do this before trying the fragment fit because any peaks which are not found in any data set will be discarded and have to be re typed 14 Set the search tolerance to 500mDa 0 5Da 15 Select fit fragment calibration in the fragment calibration setup window The results of the fragment calibration will be displayed as the number of times each peak is used and the average error of the peak When complete check that 486 Axima Performance Chapter 28 Fragment ion calibration All of the fragment peaks listed in the setup window have been used in some of the fragment spectra no completely missed peaks The residuals are no more than 0 2Da ideally they will be lt 0 1Da 16 As a check if fragment fit is ticked in the calibration window the mono isotopic mass of all the fragments peaks in any of the 10 spectra will be within 0 3Da of their theoretical value If the fragment calibration procedure has failed any of the fragments in the list or failed completely check for the following common mistakes J If the The search tolerance was not set to 500mDa half a Dalton The apparent masses are the actual frag
369. n To create a new parameter set simply enter the name for the new parameter set in the File name entry and press Save Select the Save Defaults option to save the current set of parameters into the default parameter set C Program Files Shimadzu Biotech Launchpad parameters tof_parameters It is this tof_ parameters set that is loaded and optionally saved each time the MALDI MS software is opened closed To automatically save the defaults upon closing select the Save parameters on exit option from the Configuration Environment tool see Environment Configuration Editor on page 60 Opening and saving parameter sets Chapter 10 Putting comments with collected data Chapter 10 Putting comments with collected data 84 Chapter 10 Putting comments with collected data Introduction Putting comments with collected data is a necessity which is often overlooked until sample data is reviewed at a later date Often the question How did prepare that sample or What matrix was used could easily be answered if informative comments were available with the data This facility has been provided in the form of a Comments window The Comments window is available by selecting Comments from the File menu This window Figure 10 1 allows any comments to be typed in and stored with the data angio2_msms0006 p14r_msms0004 MALDI MS SE Eg File Edit View Instrument Automation Processing Help Open tHo kje Dis
370. n allows such filters to be saved to or loaded from ASCII text files Two main filter categories can be defined namely relative and absolute peaks To define an absolute peak to be filtered simply enter its mass all peaks located within the tolerance window about this mass will be filtered Additionally all peaks less than or greater than an absolute mass can be filtered by preceding the Filtering specified peaks Chapter 16 Cleaning up data mass with either the lt or gt characters respectively Relative peak filters are entered by preceding the mass with either a or character for negative or positive relative filters respectively Thus a relative filter of 23 will filter all peaks which occur within tolerance at 23 Daltons higher than any other peak in the list It is possible to enter a chemical formula in place of a mass thus 23 above could have been entered as Na To place a new entry in the list box select Insert to create a blank entry then simply click the mouse in the new blank entry and key in the filter e g 23 The filters defined in Figure 16 12 above will filter all peak less than 5670 Daltons and all greater than 5810 Daltons Additionally any peak at 5720 6 0 5 Daltons will be filtered Finally any peaks found 74 0 5 Daltons above other peaks will be filtered This is illustrated in the spectrum in Figure 16 13 below Filtering specified peaks 261 Chapter 16 Cleaning up data pli
371. n standard ASCII character set and the default file extension is expected be txt Select Import from the File menu as shown in Figure 42 8 Sequence Calculator OF x File Edit View Settings Import Sequence 1 Lx Look in e Peptides x ck EA My Recent Documents Desktop My Documents g or My Computer File name l x Wolter Files of type Text files txt x Cancel Places I Open as read only Figure 42 8 Import Sequence window Introduction 609 610 Chapter 42 Sequence Calculator Select the required folder and the filename and press the Open button The text file will be read and any characters in the file will be interpreted as amino acids If a character is not present as a short symbol in the amino acid database it will not be included in the sequence If any characters are not recognised a complete list of all omissions will be displayed in the Log Window The sequence will be terminated when the end of the file is reached NOTE that the imported sequence will be inserted into the displayed sequence at the current cursor position s This allows sequences or parts of sequences to be added into other sequences to make larger peptides If the imported sequence is to be a completely new sequence then select Delete sequence from the Edit menu prior to importing the sequence This will delete the sequence in the current viewing panel Loading previously created se
372. n has been provided to allow the calibration to be forced to Fit through zero zero mass at zero time This effectively provides an extra reference point allowing a calibration to be performed with only one calibrant reference However where possible a minimum of two reference masses should be used The tolerance window can be applied to the spectrum trace cursors by pressing the apply to cursors button t This will cause tolerance cursors to appear around the range cursors so that the positions of reference peaks in the spectrum can be monitored If the tolerance window appears to be too small it can be increased on the Calibration window and vice versa Figure 27 8 100 5599 93 Tolerance x 2 804 5557 10 5643 43 cl wia 404 204 5560 5580 5600 5620 5640 5660 5680 Mass Charge Figure 27 8 Using tolerance cursors in calibration Assuming that the reference peaks are within the tolerance window chosen an automatic calibration can be carried out by pressing the Calibrate button If the calibration fails peaks may be outside the tolerance window then reference peaks can be marked manually using the cursors If the calibration failed then press the Undo button This has the effect of re displaying the data using the previous calibration Calibration of the instrument 470 Chapter 27 Instrument Calibration Assigning reference peaks using cursors When peaks are not close t
373. n multiplier lt Tolerance unsigned integer gt lt ToleranceUnits enumeration gt Da mDa ppt ppm lt NoiseEstimate enumeration gt Median Baseline lt LowerLimitAreas unsigned integer gt 0 100 percent modifications as of primer lt LowerLimitSN unsigned integer gt applied to the primer mass lt LowerLimitRes unsigned integer gt applied to the primer lt UpperLimitAreas unsigned integer gt 0 100 percent modifications as percent of primer lt UpperLimitSN unsigned integer gt applied to primer mass lt UpperLl mitRes unsigned integer gt applied to the primer lt GenerateMetafiles enumeration gt true false lt PrintS pectra enumeration gt true false lt OverwriteFiles enumeration gt true false lt LowerDisplayLimit unsigned integer gt percent lower mass used minus percantage lt UpperDisplayLimit unisgned integer gt percent upper mass used plus percentage lt OutputFilename string gt destination of results Multiple ModificationEntry blocks are allowed ASCII Text Method file format Chapter 15 Automated operation Block lt MSx gt lt Output gt lt Print gt lt PrintTime enumeration gt AfterAverage EndOfSample lt PrintFormat enumeration gt Colour Monochrome lt PrintFontScale unsigned integer gt 1 100 percent lt LeftMargin float mm gt lt RightMargin float mm gt lt TopMargin float mm gt lt BottomMargin float mm
374. n on the Chromatography window Chromatographic peak detection Chapter 31 Starting another data processing window Chapter 31 Starting another data processing window 2513 514 Chapter 31 Starting another data processing window Any number of MALDI MS programs can be started at any time however only one of these can be used to control and collect data from the instrument The only restriction will be the amount of memory available on the host computer The new base window will appear with all options relating to data collection disabled The controls on this new window are otherwise exactly the same as on the original window Note that when you have more than one base window present each base window can have its own sub windows so for example you may have two Peak clean up windows shown at the same time You may find it more convenient to only have one MALDI MS program shown at a time If you close the base window all of the sub windows associated with it are closed at the same time and the window s icon appears on the Taskbar When this iconised window is re opened maximised all of the sub windows will re appear in their previous positions on the screen Chapter 32 Archiving data Chapter 32 Archiving data 516 Chapter 32 Archiving data Programs Favorites Documents we fe ee Settings a Introduction SGi Shimadzu Biotech Launchpad ae Element Datahese Editor
375. n the last amino acid in the sequence the whole region selected will be highlighted Alternatively to select a small region within the viewing panel click SELECT on the first amino acid and drag the mouse while holding down the SELECT button over the amino acids to highlight Finding a specific sequence Occasionally you may wish to locate a specific group of amino acids sequence tag within a much larger sequence Select Find on the popup Edit menu and enter the required Chapter 42 Sequence Calculator sequence into the Find Sequence window Figure 42 13 and press the Find button If a matching sequence is found it is displayed in the edit window in inverse video Sequence Find and Replace Find PPR Replace PPA Replace Replace All Found at position 13 15 Figure 42 13 Find sequence window Each press of the Find key will find the next occurrence when the end of the sequence is reached the search will repeat from the start again Protecting groups Protecting groups can be attached to specific sites within the sequence To attach a protecting group first highlight a single unit within the chain as shown in Figure 42 14 then select Protecting groups from the popup Edit menu E E ME e Protecting Groups x GLYAPSHLVLTMGHGNS Acetyl i Acetamidomethyl 5 10 15 Adamantyloxy Benzyloxycarbonyl t_Butoxycarbonyl i i 7 benzyloxymethyl Highlight a single unit and t_Butoxyne
376. nalysis using Mascot search engine Other database searches You can also search on the following databases peptident used for PMF MS searches profound org profound mono You can use the subsequent forms within the Internet Search window to prepare your data However as MALDI MS does not support these products we cannot guarantee that the data you enter will transfer correctly Use the procedures described in the two previous sections as a guide Other database searches 315 Chapter 18 Protein peptide analysis using Mascot search engine 316 Other database searches Chapter 19 lon finder Chapter 19 lon finder 318 Chapter 19 lon finder Introduction Introduction You can define a list of mass peaks of interest and use the Ion Finder feature to extract the corresponding intensities from a spectrum The feature allows you to import or generate a list of masses tolerances export the data for use in third party applications For each peak of interest lon Finder examines the spectrum and extracts its intensity mass area The results are presented in a text report You can also save and load lon Finder settings Peak intensity The intensity of a peak is defined as the sum of the intensities of the mass spectrum trace between the start and the end of the peak Tolerance The tolerance is measured is about the mass For example if the mass is 1000 and the tolerance i
377. nd HPLC elution coefficients Extensive editing facilities for modification of protein structures Rapid molecular weight and elemental formulae calculations from a user definable amino acid database Entering a new sequence To enter a new sequence into the Sequence Calculator click the mouse Select button in the top panel so that a triangular insertion cursor is displayed s Type in the amino acid sequence as single letter mnemonics as shown in Figure 42 3 Any characters which are not recognised will generate a warning message Sequence Calculator Oy x File Edit view Settings a PPPPPPPPPPPPPPR 5 1 us 44 4 PPI Synchronise Name Unte po a N terminus Hydrogen H Monoisotopic x C terminus Hydroxy H O Y Fragmentation Singly charged Y Generate Peak Matkers Calculate Cation Proton H iw Digest I Length 15 Figure 42 3 Entering a new amino acid sequence Introduction 603 604 Chapter 42 Sequence Calculator Introduction Amino acid notation Amino acid sequences can be entered using the Keyboard tab of the Peptide Settings window Figure 42 5 or the computer keyboard The Peptide Settings window is obtained from the View menu as shown in Figure 42 4 Sequence Calculator File Edit View Settings a PPPPPPPPPPPPPPR 5 1 15 Figure 42 4 Starting the Peptide Settings window The Keyboard tab shows a button or key for each amin
378. nd then changing the value in the dialogue that will appear If the precursor selection resolution and CID waveform amplitude have not previously been set or require modification this should be done using the QIT ToF MS tab of the acquisition window described in Setting up MS parameters in the Axima Resonance on page 137 The instrument is now ready to acquire data as described elsewhere in this chapter 162 Acquiring MS MSn data on Resonance Chapter 14 Collecting data from a sample lon gate accuracy The ion gate filters out unwanted ions and only allows the required ions to remain in the ion trap for manipulation You set the ion gate width using the QIT ToF MS tab and the Resolution field 4 Acquisition positive Firing Exp Tech Auto Quality Storage Slide Raster Tuning QIT ToF MS 2 3 MS MS Precursor Ion 1046 540 AE Precursor Ion 931 530 t Resolution Wide Isotope Selection 70 X Resolution Standard Resolution i CID Control CID Control Soft hard Soft Hard E A E EEA ai 0 btn R AA 200 5 5 20 5 Adjust Gate Clear All Apply The table below gives the approximate ion gate widths for different precursor ion masses Precursor ion mass Resolution 500 Da 1 000 Da 2 000 Da Wide 70 7 Da 14 Da 28 Da Std 250 2 Da 4Da 8 Da High 500 1 Da 2 Da 4 Da Extra high 1000 Y Da 1 Da 2 Da However the ion gate may drift by 0 5 Da due to the high voltage and RF circuits wa
379. ndows event viewer 70 Chapter 7 The Log Window Events logged Event categories The Axima can generate up to five categories of event messages Information e Warning e Error Audit success Audit failure You can be filter these messages using the Event Filters window 1 From the MALDI MS main window select View gt Events Logged Events Logged Eg Select which types of event you would like MALDI MS to log You can view this log using the system Event Viewer and optionally notify a number of recipients via e mail IV Log warning events IV Log information events T Log audit success events T Log audit Failure events Use of e mail is currently disabled because there is no MAPI e mail client running or it has been incorrectly configured ema Cancel If the error message shown above is displayed the Email feature is not available If you require this feature a Click the Cancel button to close the Events Logged window b Open a MAPI client e g MS Outlook not Outlook Express c Open the Events Logged window again View gt Events Logged and the Email button is available 2 Tick or untick the required categories Under normal circumstances information messages only are seen and can usually be treated as progress reporting Warning messages are usually seen in Automatic mode and Events logged Chapter 7 The Log Window indicate a problem that was recovered from for e
380. nel are available The instrument must not be moved while switched on as doing so may result in damage to the vacuum pumps Safety statements and warnings Chapter 2 Safety warnings Sample records A record should be kept of all substances analysed with the instrument and their concentration level amounts This record will be needed by the Kratos Analytical Service Centre should any part of the instrument need servicing replacing Any item returned for repair replacement must be accompanied by this sample report and a completed copy off the Returned Equipment Declaration with reference to the Equipment return declaration procedure which may be found in the Customer support guide Guidelines Take note of the following guidelines e Your equipment is uncontaminated if it has not been used or if it has only been used with substances that are not dangerous Your equipment is contaminated if it has been used with any dangerous substances if your equipment has been used with radioactive micro biological substances or biologically substances it must be decontaminated using an approved decontamination process before an engineer at any Kratos service centre can proceed with any repair or service You must supply independent proof of decontamination for example a certificate of analysis to your supplier with the declaration Contact your service centre for advice We recommend that contaminated equipment be transported
381. nfidence instruments provides pulsed extraction for improved sensitivity and resolution However this technique gives rise to a nonlinearity in the theoretical TOF law of calibration which is not handled by the standard method of calibration This effect is quite small and is not noticable unless the data is high quality and from a large mass range Since the Axima Assurance and Confidence can easily generate such data the software provides a means to correct for this effect The correction will be automatically applied if a calibration is performed on 3 or more data points In this case the correct option on the calibration window see Figure 27 1 will be enabled and automatically selected When calibrations that have correction turned on are saved then these calibrations automatically contain the information necessary to apply the correction to other data even if a simple 2 point calibration is made later The correction factor itself is dependant on the ionisation parameters in the source The most dominant of these is the matrix being used though the analyte class also has an effect Therefore different calibrations and correction factors are required for different matrices and ideally for different classes of analyte compounds polymers peptides oligonucleotides etc When using this feature bear in mind that the quality of the data is very important If a small number of poor quality peaks are used for calibration then it is possib
382. ng gt lt Acquisition gt lt Processing gt lt Calibration gt lt PeakCleanup gt lt MonolsotopicPicker gt lt PeakFiltering gt lt Mascot gt lt Processing gt lt Applications gt lt CompareSequence gt lt OligoAnalysis gt lt Applications gt lt Output gt lt Print gt lt Export gt lt Output gt lt MS1 gt lt MS2 gt lt Acquisition gt lt PeakSelection gt lt Raster gt lt AutoQuality gt lt LaserFiring gt lt Acquisition gt lt Processing gt lt PeakCleanup gt lt Monol sotopicPicker gt lt PeakFiltering gt lt Mascot gt lt Processing gt lt Output gt lt Print gt lt Export gt lt Output gt lt MS2 gt lt Method gt lt Group gt lt Experiment gt Values that are not specified in the experiment method will be set to default values These values are not fixed and may vary from release to release Hence ALL important values should be set ASCII Text Method file format 215 216 Chapter 15 Automated operation Block definitions for the method block are as follows Block lt DataStorage gt lt NumberOfAverage unsigned integer gt lt StoreProfiles enumeration gt Never All AfterAverage AtEndOfSample lt CompressData enumeration gt false true lt DataFile string gt Block lt MSx gt lt Acquisition gt lt Raster gt lt Centrex float microns gt lt CentreY float microns gt lt Width float microns gt lt Height float microns g
383. nge 10 10000 0 Calibration pooo El WEY Applications Laser Firing Me Compare Se p E v 7 ES 1 Oligo Analys Power 90 m B M Output Mc Print wer 110 Ve Export Profiles per samp MU Report Via lon Finder Shots j100 7 accumula a ME MS MS Ele MEQ Acquisition SE Peak Select BH Raster MX Auto Quality Me Laser Firing a ME Processing EZ Peak Clean MIME Monoisotopi MEX Peak Filterin ME Mascot X gt Data Storage Get Manual Settings Sa Figure 15 1 Starting the Method Editor window Laser Rep Rate 50 0 Ion Gate _off on Blank 5 M P Ext optimise For fu I Restrain peak cleanup 166 Introduction Chapter 15 Automated operation Method Editor The Method Editor Figure 15 1 provides the facility to edit load and save methods which can then subsequently be applied to samples in the Auto Experiment application see Auto Experiment on page 203 A particular application of these methods is to perform a PMF Peptide Mass Fingerprinting experiment A Method is a collection of parameters used in the analysis of one or more samples A Method defines how a sample is to be Acquired how the acquired data is processed and the format of any results to be obtained from the data The structure of a Method is defined in a simple tree structure as shown in Figure 15 2 Sample Method default mtd My Parent Node selected for AY Acquisition current method
384. nge of profiles and mass range are entered as for the spectrum display As with spectra it is not necessary to enter a sample number or range of profiles before collecting new data as these will be updated automatically Displaying Chromatograms 281 282 Chapter 17 Viewing the collected data Finding the sweet spot using chromatograms There will be a significant rise in signal intensity where the largest number of ions are obtained on the sample spot Figure 17 9 t 182 mV mass 5500 to 7000 Smoothed 1 Figure 17 9 A chromatogram display with sweet spot marked The instrument can be set to collect data only from the sweet spot by setting the Aim on the Laser Firing window to the start and end position of the sweet spot To do this place the range cursors on a chromatogram display at the start and end profiles of the sweet spot Press and hold down the mouse ADJ UST button on the chromatogram display A vertical cursor will appear Position the cursor at the start profile of the sweet spot and release the ADJ UST button Repeat this procedure to position a cursor at the end profile as in Figure 17 9 and press the 4 cursors button on the Laser Firing window aim line This will set the laser position for future laser firing to be the range between the two cursors The Aim will be updated to show the selected range Pressing the 1 gt button sets the Aim back to the full range 0 1000 When data ha
385. ning file default_linear Load Save r Voltages m PE settings gt Source tat rea Source X defl 100 Scaling factor 50 0 Einzel Lens 6250 3 Source defl wo Switch delay po m Pulsed extraction 2000 Ej Gating Reflectron 0 3 Refl X defl 0 3 Scaling Factor 10 Linear detector 2600 3 Refl defl 0 4 Offset 3 Refl detector 0 Reflectra Print Adjust Figure 13 1 Tuning window Axima Performance At the top of the window are the Load and Save buttons which provide access to the standard dialogues for loading and saving tuning files The parameters are grouped into three sections these are Voltages PE settings and Gating There are default files for tuning the instrument in positive and negative linear and reflectron mode itis strongly recommended that these files are not altered other than by an advanced user The Adjust button is for service engineers only It is a toggle switch which is intended to prevent accidental changing of parameters which are critical for optimum instrument performance once a tuning file is loaded the values can only be viewed unless the Adjust button is depressed To discourage users from altering the tuning parameters for an experiment it is password protected Tuning for an acquisition 35 136 Chapter 13 Preparation for data collection Axima Resonance model As for the other Axima instrume
386. nly the first instance of the well is processed remaining instances are ignore or skipped The list control is used to display current groups select method files select data files set sample comments and modify selected samples Table 15 8 Auto experiment samples icons Create and append a new sample group to the list control The samples in the group are the ones that are currently selected in the Selection plate Auto Experiment 207 208 Chapter 15 Automated operation Table 15 8 Auto experiment samples icons Modify Update the currently selected sample group with the currently selected wells in the Selection plate Remove the currently selected sample group Move the currently selected group up the list This results in the selected group being processed sooner than it would have been prior to the move Move the currently selected group down the list This results in the selected group being processed later than it would have been prior to the move Ihe list control can be used to display and modity sample group settings Some of the columns in the control are editable fields and can be modified Double clicking the left mouse button on the required cell displays an edit box that is used to enter new values Moving to a different field or pressing the Enter key will update the field with the new value If a field displays a button then a further dialog box will be activated when clicking On it the type of d
387. nnotation window displays two types of labels Manual and Automatic labels Automatic labels are generated by the software and cannot be edited Manual labels are created by the user and can be edited The list in the window displays all labels which match the settings of Type Dataset and Trace choose the setting accordingly and any labels matching the selections will be listed The list can be sorted in either I ncreasing or Decreasing mass alphabetic order or unsorted Annotation 391 392 Chapter 20 Managing Data Displays Annotation Any labels in the list can be deleted by clicking the mouse MENU button over the list of annotation symbols The menu which appears allows deletion and removal hiding of annotation symbols to be performed Figure 20 44 Hide selected labels Show selected labels Hide all labels Show all labels Delete selected labels Delete listed Delete listed between cursors Delete all labels New Figure 20 44 Annotation menu To delete labels in the list select the entries to be deleted and select Delete selected labels from the menu Multiple entries can be selected and all labels in the list can be deleted by selecting Delete listed or alternatively all labels in the selected display can be deleted by selecting Delete all labels To delete selected labels from a graph place the range cursors so that they enclose all of the cursors to be deleted and press Delete listed between cursors Lab
388. nt Automation Processing Help lnt glij e 2 a x lt le Display Speci Profiles fi p Massesi 515501 iel 506 33 525 500 550 600 2 e 4 Mass Charge Mass Charge int int 522 03 100 100 80 a 2 c int 1 c F1 60 40 519 86 5 2 03 451 94 49209 49 05 20 3 07 o 17 2 c o 450 500 550 1 c F1 515 520 525 530 1 c F Mass Charge Mass Charge 4 if m For Help press F1 dM 68 34 M dM 7 40 Figure 20 24 Copied display Inserting displays Displays can be copied into another display to create inset displays This feature can be used for publications to show a specific region of a graph as an inset It can also be used during data collection to provide an inset view of the progress of data collection from a given sample spot Creating inset displays Select the display to be used as an inset Position the range cursors on the display to contain the inset so that the cross hairs mark the boundary of the inset Press the keyboard Shift key and hold it down while clicking on the toolbar copy display button al The selected display will be made into an inset between the cursors and the range cursors will be removed Figure 20 25 Copy insert and delete displays 367 Chapter 20 Managing Data Displays ff angio2_msms0006 MALDI MS Of x Eile Edit view Instrument Automation Processing Help i sja e 2 Bl X E Disp Spectum T Profiles f1 Masse
389. ntly displayed Method can be saved to file by selecting the Save Method button as shown in Figure 15 3 below The default method file extension is mtd When saving a Method every parameter will be saved to file regardless of the node selection state Save Method ae Save As 21 x Save in E Methods x ae My Recent Documents Desktop Sequence Report ti default mtd E LC MALDI Calibrant mtd E LC MALDI Sample mtd 2 My Documents i Sr My Computer lt cine File name default mtd bi Places Save as type Method Files mtd x Cancel Figure 15 3 Save Method window Chapter 15 Automated operation A previously saved method can be loaded into the Method Editor by selecting the Load Method button as shown in Figure 15 4 below Load Method e Open 7 x Look in E Methods x ce Fe Sequence Report ee E default mtd My Recent Documents Desktop My Documents PE My Computer E LC MALDI Calibrant mtd E LC MALDI Sample mtd m ets File name l z Open Places Files of type Method Files mtd x Cancel Figure 15 4 Load Method window Data Storage The Method Editor Data Storage window is identical to that described in Storing collected data on page 146 To access the Data Storage window select the Data Storage button as shown in Figure 15 5 basso o e Average fi a profiles Store profiles fat end o
390. ntly loaded dataset and add them to the Mascot search You specify the mass range as a percentage of the parent mass i ees of 40 Segments 5 Low peak High peak ae 2 A 98 percentage limit percentage limit Fetch Peaks Title Parent 1722 958 Da Parent mass 1722 958 Parent massis Monoisotopic C Average Mass a for Intensity aare 2 246 503 3017 99 z 261 128 2664 81 x 261 363 2412 21 eB 261 754 2367 79 w amp aoc 270 7677 79 i Delete Peak ae eed The value in Parent mass field is fixed However you can change the other values a The Title field is based on the parent mass You can change this field if required b You can set whether the precursor parent ion mass is monoisotopic or average Click the required radio button The Mascot search engine requires that you use I gt all monoisotopic masses or all average masses but not a mixture To satisfy the requirements of the search engine the MALDI MS software will use the average precursor parent ion mass even though you have selected it as a monoisotopic mass c The Mass and Intensity fields are populated with peaks extracted from the spectrum You can delete a peak select it and click the Delete Peak button 310 Mascot MS MS searches Chapter 18 Protein peptide analysis using Mascot search engine 4 Click the Add button the peaks are added to the Mass list Mass list This section allows you to specify the list of masses on whic
391. nts a tuning window is provided for the Axima Resonance instrument which allows experienced users to tune the instrument Acquisition positive Firing Exp Tech Auto Quality Storage Slide Raster Tuning QIT ToF MS Tuning file Voltages Intro Endeap 4 Yy Reflectron Centre Y lon Polarity Save Extr Endcap TF jhi Reflectron Back av ToF Float y Detector v Print Adjust Figure 13 2 Tuning window Axima Resonance One of the main advantages of the Axima Resonance instrument is that the ionisation parameters are decoupled from the mass analyser by the ion trap This is reflected in the tuning tab which allows only the voltages associated with the analyser to be tuned The tuning of the source conditions have been separated out and placed into the experiment flowchart editor with all of the other parameters associated with the introduction of ions into the trap This means that under normal circumstances there is even less reason to adjust these values The Adjust button is for service engineers only It is a toggle switch which is intended to prevent accidental changing of parameters which are critical for optimum instrument performance once a tuning file is loaded the values can only be viewed unless the Adjust button is depressed To discourage users from altering the tuning parameters for an experiment it is password protected Tuning for an acquisition Chapter 13 Preparation for
392. nu presents the Delete menu This menu allows a Row containing the selected display or a Column containing the selected display to be deleted The Multiple displays Chapter 20 Managing Data Displays displays to be deleted will be highlighted and a confirmation message will be presented If accepted the highlighted displays will be deleted Tile Manager Operations in and can also be achieved using the Tile Manager which provides a visual representation of the current tile layout and an interface to select insert and deleted tiles See Figure 20 7 Left mouse button double clicking in the current tile layout area will set that tile as the active tile When the mouse pointer is over a menu item that can be accessed it becomes highlighted Clicking on a highlighted menu item will cause that operation to be carried out Tile Manager x Current Tile Layout Cancel Insert Row Column Inset Display Settings Delete Row Column Inset All Insets Figure 20 7 Tile Manager Enlarging displays Displays can be enlarged to full size full width height full screen or reduced back to normal using the Displays toolbar zoom options Displays which are enlarged will cover up other displays The covered displays will reappear when the enlargement is cancelled It is not possible to have a mixture of full width and full height displays as this would cause overlap of the displays to occur Multiple displays 345
393. o acid in the amino acid database see Defining amino acids on page 574 Pressing a key on the tab with the mouse SELECT button enters that amino acid into the sequence shown in the editor panel Alternatively sequences can be entered directly from the computer keyboard by typing the letter corresponding to the amino acid short symbol Short symbols nomenclature for amino acids i e single letter mnemonics are currently more popular than the previous three letter nomenclature However to accommodate individual preferences single letter multiple letters and full name referencing is permitted within the program Any amino acids defined using the Compounds Editor will appear as keys on the Keyboard tab These can be defined at any time as soon as they are entered into the Compounds database they will be displayed in the Peptide calculator Chapter 42 Sequence Calculator A e cl of e E e 2 x L Ml nj of P o r s x w x z Short format Alanine Arginine Asparagine Aspartic_Acid Cysteine Cysteine_oxidised Glutamic_acid Glutamine Glycine Histidine Isoleucine Leucine Lysine Methionine Ornithine Phenylalanine Phosphoserine Proline Serine Threonine Tryptophan Tyrosine Valine Long format Full format Figure 42 5 Keyboard tab of the Peptide Settings window The type of Keyboard tab displayed is controlled by the Keyboard option on the Display tab of th
394. o the expected positions they can be mass assigned using cursors Any number of peaks may be marked in this manner and the calibration will be extrapolated over all data collected To mass assign the peaks using cursors 1 Collect data containing the calibrant reference peaks Set Traces to Processed on the Display contents window 2 Display a spectrum of the collected data and set the displayed mass range to the full range 3 Select a Tolerance of 5 Da and press the t button 4 Insert another display above the current one by using the Display menu and selecting Insert gt Row Click the mouse SELECT button in the newly created display 100 Yolnt 100 92 m sum 9388 mV Profiles 0 100 Smooth Av 10 Baseline 40 268 94 356 94 1170 60 1690 96 2734 08 3367 01 3947 76 1000 2000 3000 4000 Mass Charge Figure 27 9 Setting up the first display for calibration 5 On the second display select a mass range of the order of 20 Da around the reference peak Do this by expanding a region containing the reference peaks to be used in the calibration by dragging the mouse whilst holding down the mouse SELECT button 6 On the first display showing the full mass range hold down the keyboard Ctrl key while clicking the mouse SELECT button over the reference peak The second display will show the reference peak in a 20 Da Calibration of the instrument Chapter 27 Instrument Calibrat
395. oF 2 7 0 20051223 Mode reflectron Power 62 Gate 1515 00 1555 00 Shimadzu Biotech Axima ToF 2 7 0 2005122 lnt 15 m surm 349 mv Profiles 1 23 Smooth Gauss 20 Baseline 50 lnt 167 miv sum 3842 mv Profiles 1 195 0470 r323 1633 8580 r2193 100 100 1341 2362 1545 1534 8621 12739 90 90 Wi 80 804 70 704 60 60 so 6 0995 r 179 50 a 9512862 313 40 1342 6 404 2 2003 r544 1145 5434 r465 30 30 j 1146 8715 1343 20 536187 13 r364 10 at ld a MAMI daital a ee 200 400 600 800 1000 1200 1400 1530 1535 1540 Mass Charge Mass Charge Table 28 5 Actual masses P14R MS MS spectrum with the fragment calibration applied Data p14r_msms0004 13 c 28 Sep 2005 13 35 Cal fc_test 28 Sep 2005 13 34 CID Data p14r_msms0004 3 c 28 Sep 2005 13 Shimadzu Biotech Axima ToF 2 7 0 20051223 Mode reflectron Power 62 Gate 1515 00 1555 00 Shimadzu Biotech Axima ToF 2 7 0 20051 22 lnt 15 m sum 349 mY Profiles 1 23 Smooth Gauss 20 Baseline 50 lnt 167 m sure 3842 mv Profiles 1 525 7 B00 rB96 1 1533 8580 r27 64 100 100 E 1406 67 1 18 1534 52 1 12739 90 90 80 80 70 70 60 60 a 2 1536 8665 r2652 40 1407 6386 r4t 40 E 1274 1723 r751 30 9 30 20 10 i 0 1000 1200 1400 1530 1535 1540 Mass Charge Mass Charge Axima Confidence Chapter 28 Fragment ion calibration Table 28 6 Apparent masses the same P14R MS MS spectrum without the fragment
396. ocessed trace The lower trace is the Processed trace which shows the peak limits for each detected peak in the spectrum Yolnt 100 5590 5600 5610 5620 5630 5640 5650 5598 7 5590 5600 5610 5620 5630 5640 5650 Mass Charge Figure 20 33 Baseline and Peak Limits on a graph Customising Graphical Reports 377 378 Chapter 20 Managing Data Displays Graph Scaling The individual data profiles can either be scaled to the largest peak in the window Automatic which is the normal mode of operation or can be set to scale to a user defined value Using Profile Scale Automatic the data is normalised to the largest peak in the currently displayed mass range which will be assigned 100 intensity all other peaks will be scaled relative to this one Using Profile Scale Manual allows the user to specify the maximum value on the Y axis in millivolts thereby showing all profiles in the display relative to that value Using Profile Scale Relative to allows the user to specify the loaded dataset to which all other displayed profiles should be scaled If that dataset had a maximum displayed Y axis value of 50mvV then all displays would be shown normalised to the same scale At the top of the graphs the graph title indicates the value to which the traces are scaled in the same colour as the trace itself and in the order in which the datasets were loaded When collecting data it is normal to have the graphs scaled auto
397. oduction to the MALDI MS software Chapter 3 Getting started If another MALDI MS program is started while the first one is still running an error will be reported in the Log window and the new window which starts can only be used to process data which has already been collected On such windows any instrument control menus will be unavailable for selection appearing as grey options The base window has five menu items marked File Edit View Instrument and Processing These buttons are arranged from left to right in the approximate order in which they will be used to collect and process data from a sample Clicking on a menu option with the mouse SELECT button displays the respective menu from which further selections can be made Figure 3 5 x angio2_msms0006 MALDI MS File Edit View Instrument Automation Processing Help Display Spectrum be Save As Open Auto Experiment Results Comments Parameters Print Ctrl P Print Preview Print Setup Figure 3 5 Making menu selections from the base window To obtain help at any time click the MALDI MS toolbar help button k2 position the mouse help pointer R over the item for which help is required and click the mouse SELECT button Alternatively press the keyboard F1 key or select Help topics from the MALDI MS help menu in both cases the MALDI MS Help topic viewer will be displayed Introduction to the MALDI MS software 29 Chapter 3 Getting
398. ofiles Apex mV Profiles Masses 101 250 11250 751 1000 11100 11549 1301 1450 11100 1651 1900 11100 Peak display Previous First Last Clear 4 peak s selected Figure 30 4 Peaks detected in the Chromatography window The table of detected peaks can be ordered in Ascending or Descending Height Mass or Profiles This flexibility allows the table to be re ordered depending upon individual requirements One user may place emphasis on reporting the apex position of the detected peaks whilst another may need to know in which profile ranges i e where on the slide the peaks occurred The table not only serves as a scrolling list of the detected peaks but also as a means of viewing the detected peaks For example on the base window set Display to Chromatogram When an entry in the Peaks detected list is selected using the mouse SELECT button the chromatogram for that peak will be displayed The Update option determines which parameters on the base window will be updated when the peak is selected The Profiles Masses or Both profiles and masses can be updated for each peak Having selected an entry in the list press the Next button repeatedly to step through the list displaying the next peak The other options are to display the Previous peak First peak or Last peak the base window selected display will be updated each time the button is pressed To clear all peaks in the list press the Clear button
399. oid vE MS MS J MEY Acquisition fe Peak Selection v Raster Ea p zd inite V8 Laser Firing ee h vI Processing EK Peak Cleanup VI Monoisotopic Peak Picker VEA Peak Filtering VS Mascot My Output Data Storage Get Manual Settings Save Method Load Method Figure 15 12 Method Editor Peak Cleanup Gradient Centroid Peak Detection Settings Set up the peak cleanup parameters as described in Cleaning up data on page 235 176 Method Editor Chapter 15 Automated operation The peak cleanup parameters will always be applied to the current method as indicated in the tree by the absence of the selection box Peak Picker The Method Editor Monoisotopic Peak Picker window provides functionality for the selection of monoisotopic peaks from a peak envelope particularly in the scope of a PMF experiment when importing acquired peaks to a Mascot search To access the Monoisotopic peak picker window select the Monoisotopic Peak Picker label from the tree as shown in Figure 15 13 below Method Editor default mtd Sample Method Sample Method default mtd Monoisotopic Peak Picking iy Parent M I Acquisition 2 a eae Method Poisson peptide VIe Laser Firing VY Processing VIL Calibration EZ Peak Cleanup Ali Pe Minimum mass 0 Maximum mass v EN Peak Filtering Minimum isotopes VS Mascot I Applications Maximum intensity variation Compare Sequence Oligo Analysis IV Overlapp
400. olding down the keyboard Shift key while pressing down the mouse SELECT button with the mouse pointer within the selected display The peak in the selected display nearest to the mouse pointer will jump under the mouse pointer still holding down the mouse SELECT button the peak may be repositioned within the display the Shift key may be released as soon as the mouse button is pressed down Panning using two displays Create two displays within the main window In one of them select the full mass range and in the other select a delimited mass range of say 1000 daltons Select the display with the delimited mass range and in the other display with the full mass range hold down the keyboard Ctrl key while clicking SELECT with the mouse As the mouse is dragged in this display the selected display will show the delimited mass range e g 1000 daltons centred on the current mouse cursor position Panning displays 395 Chapter 20 Managing Data Displays File Edit view Instrument Automation Processing Help gja sael 2 lt Display Spectrum Profiles fi Masses 1022 1080 I gt 6 7 mV sum 955 m Profiles 1 142 Smooth Av 20 Baseline 80 Press Ctrl and drag the mouse in to this display selected display will pan following the mouse 1534 33 4000 80001 c I Mass Charge 0 5 mV sum 69 mV Profiles 1 142 Smooth Av 20 Baseline 80 1047 46 Selected display 1030 1050 1055 1060 1070 1075 Mass Charge
401. on the Archive to file window is displayed as shown in Figure 32 5 Archive to file 12 x Save in 6 My Documents gt ek EJ My PSP8 Files Save as lype Zip file zip x Cancel Figure 32 5 Archive to file window for archiving Navigate to the required storage media type in a name for the archive file and press Save For all types of removable media a message will appear DynazIP ZIP Request x Please insert the first disk of the Multi volume set Press OK when ready or Cancel to abort H Cancel Simply ensure that the selected media is in the drive and press OK or hit Return A progress indicator will be displayed and will indicate the progress of the archive to the selected media Figure 32 6 It shows a count of the number of items to archive the current item being archived and the percentage completed Item 171 of 303 al J 56 SHG DREBBROREL Figure 32 6 Archive progress indicator Introduction 519 520 Chapter 32 Archiving data At any time during the process of archiving files the Cancel button on the progress indicator window can be used to stop the process Should further removable media volumes be requested a message to that effect will be displayed and the next volume in the series should be inserted The archiver is unaware of any changes made to the contents of any fixed drives or network drives For this reason if drive contents change or new data is collec
402. on a single graph trace on each display or across all displays Cursor height Cursors lnt 100 522 03 Figure 20 20 Cursors set to graph height Chapter 20 Managing Data Displays lnt 100 80 60 404 20 0 lnt 100 80 60 40 204 Figure 20 21 Cursors set to display height When two or more displays are stacked above each other showing the same data range e g when comparing data from different sample spots it is sometimes useful to highlight features at exactly the same point on the mass scale in both graphs Cursors 363 364 Chapter 20 Managing Data Displays Cursors lnt 100 50 lnt 100 50 lnt 100 50 lnt 100 50 To do this set the cursor Height option to Window The cursors will now be drawn through all stacked data displays 440 460 Figure 20 22 Cursors set to window height Chapter 20 Managing Data Displays Copy insert and delete displays Copying displays Displays can be copied from one place to another within the same window Select the display to be copied then press and hold down the mouse SELECT button on the Display toolbar copy display button al The mouse pointer will change into a copy display pointer and an outline will appear around the selected display Figure 20 23 Still holding the mouse SELECT button down drag the outline of the selected display to its destination
403. one terminus It is common to use a carboxypeptidase enzyme digest attacking at the C terminus end of the peptide chain The procedure is then to compare the PSD spectrum of the original undigested peptide with that after enzyme treatment i e the peptide less one amino acid In principal subtraction of the two spectra should yield information on the identity of the cleaved amino acid The analysis can then be repeated for several amino acid units along the peptide backbone studying the differences in spectra at each step The algorithm used here examines the two largest Molecular weight peptide fragment spectra and results in two types of spectra one containing N terminal ions and common internal fragment ions the other comprising C terminal fragments and internal fragment ions that were not consistent in both The second part of the procedure then uses these to calculate possible theoretical sequences which must end in the amino acids revealed by the original subtraction after carboxypeptidase treatment Chapter 42 Sequence Calculator The experiment is set up in the Nested PSD tab of the Peptide Settings window Figure 42 30 amp Peptide Settings oP x Keyboard Display Fragmentation Match Peaks Nested PSD Spectra Subtract File Parameters Start mass El End mass 1500 lM Show spectra Match tol 0 5 Da Threshold 1 mv z t 4 4 Add file Delete Clear Subtract files l Sequencing S
404. ons The v ion series is equivalent to the v ion series reported by Johnson Martin and Biemann Reference 6 The w and w ion series are equivalent to the wa and Wy ion series reported by Stults and Watson Reference 9 Spectra can consist of a series of multiply charged ions If M represents the molecular ion then a typical series of multiply charged ions might be designated by MH MH MH3 t etc The observed mass to charge ratio m z of these ions will be respectively etc Where M 1 M gt 2 M 3 M is the molecular weight of the peptide 3 terminus Hydrogen Sequence composition Elemental composition M H Average mass M H Most abundant mass M H M H Monoisotopic mass rypsin digest Average Masses M H N Position 1 L 6 2 Tos T 3 6 9 Mol Ut 712 83 175 21 288 37 Chapter 42 Sequence Calculator The Multiply charged section at the bottom of the Fragmentation tab controls the reporting of these multiply charged fragment ions The m z range entry and the Max charge entry are used to limit the mass to charge range for the multiply charged ions to be reported Setting the Fragmentation option on the Sequence Calculator window to None switches off the reporting of all PSD fragmentation For example if only enzyme digest fragments are required then using this setting only reports the digest fragments and no PSD fragmentation whatsoever Note that a digest fragments only repo
405. or a calibrant list Calibration of the instrument 473 474 Chapter 27 Instrument Calibration Saving calibrations Calibration If the calibration is acceptable and the data used for the calibration was stored then if the calibration is successful check the status bar of the main MALDI MS window the calibration will be automatically saved with the data as a cal file This means that whenever this dataset is loaded the calibration which was stored with the data will be used This calibration only applies to this dataset not to any others To use the calibration with all other data collected from the instrument after the calibration was performed then type a filename into the Name entry and press the Save button Only saved calibrations will be used when new data is collected names The MALDI MS program automatically keeps separate calibrations for each instrument mode e g positive negative ionisation modes low high mass linear reflectron A default calibration name TOF is supplied with the instrument on the computer when the instrument leaves the factory A TOF calibration is available for all instrument modes Calibrations can be saved for different ionisation modes high or low mass range and either linear or reflectron settings A name should be entered after calibration has been performed before pressing Save to create a new calibration file Should you wish to collect data using the same instrument settings and c
406. or features Select the Cursors option from the Display Options Cursors tab as in Figure 20 15 default MALDI MS Oy x File Edit View Instrument Automation Processing Help Ss kl v Toolbar lt Display Spectrum Profiles 1 Masses 12 1040 Ipi v Status Bar v Display Toolbar v Basic Parameters Display Options BEd abel contents General Graphs Graph Text Text Report Cursors Peak Labels Options Camera Type Normal E Instr t Status r r ae mae etched Fa E F Tile M vee F 3 Event ters a ra Width None Normal Display Window Height Graph Display window Join with Of Line lon Gate Colours Tl All displays OK Cancel Figure 20 15 Cursors window The Type option specifies the type of cursor to be displayed on the graphs When set to Normal the cursors appear as in Figure 20 14 The other options are Charge which can show additional cursor lines indicating multiply charged peaks or multiples of the indicated mass Tolerance which shows a tolerance band and Time which displays the sample bin value of the mass under the cursor These are explained below Cursors 355 356 Chapter 20 Managing Data Displays Charge cursors Cursors When Type is set to Charge additional cursors can be produced which show the mass positions of multiply charged fragments down to M 10 It is also possible to display cursors showing multiples of the indicated mass up to
407. or the new compound into the Name entry This name should start with a capital letter and may contain the underscore character _ as a separator but may not contain a space If a capital letter is not used for the first character of the name the program will convert the first letter of the name into an upper case character automatically For example Acetic_acid acceptable acetic_acid acceptable acetic acid spaces are not allowed in compound names Next type the empirical elemental formula for the compound into the Formula entry Spaces or full stops periods can be used in this case to separate distinct groupings For example CH3COOH acceptable CH COOH acceptable CH3 COOH acceptable Chapter 38 Creating a compound database Press OK or Apply and the new compound entry will be inserted into the database in alphabetical order The rules for entering formulae are given below Rules for entering formulae Firstly for elements from the periodic table use the elemental symbols exactly as they appear in the periodic table The number of each individual element appears after the element name e g CH3 NH2 CCl4 represent CH3 NH3 CCly To define specific isotopes use a caret e g 13C or 2H to represent 13C and 2H respectively Where a grouping of elements is repeated the group should appear in parenthesis e g CH3 CH2 9 COOH represents CH3 CH2 COOH Creating different aliases for the same compound A compound can
408. or to the next data collected Introduction 87 88 Chapter 10 Putting comments with collected data Where more information needs to be stored than the amount which can be entered into the Comments window use the Notes feature which allows text files of arbitrary length to be stored with data and is described in Creating Notes for data on page 290 Saving comments to named files It is often useful when running repetitive samples or similar samples to be able to store comments to named files This allows large numbers of comments files to be created which can then be recalled at a later data and applied to collected data All comments files are stored in the Comments folder defined in Environment Configuration Editor on page 60 within which the user can create any number of subdirectories folders To create named comments files firstly type in all of the comments required into the comments window Click on the Save file button the window shown in Figure 10 2 will appear Type in the name of the file in which the comments will be stored Select Comments File 21 x Save in E Comments x e fa My Ree Documents Desktop B angio comm s LastCommentsUsed comm D My Documents sr My Computer UMET Filename l X Places Save as type Comment files comm 7 Cancel Figure 10 2 Select Comments File window Press Save and the comments shown on the Comments window
409. ot Server This allows the FTP file transfer of Mascot result files between the Axima PC and the Mascot Server The Mascot Server must be configured as an FTP server Use the following table for guidance about the fields Table 6 1 Mascot setup fields for remote access Field Email Address Search URL FTP results server FTP results location FTP Intermediate processing location FTP config location Tick box Username Password Guidance Set the email address to which any failed aborted Mascot searches will be sent Emails will only be sent if this is defined in the Mascot config file Enter the URL for the remote Mascot search engine http www matrixscience com cgi nph mascot exe 1 Not applicable Not applicable Not applicable Not applicable Not applicable Not applicable Not applicable After making any changes click the Apply button Mascot Setup 63 64 Chapter 6 Configuring Launchpad Mascot Setup Table 6 2 Mascot setup fields for local access Field Email Address Search URL FTP results server FTP results location FTP Intermediate processing location FTP config location Tick box Username Password Guidance Set the email address to which any failed aborted Mascot searches will be sent Emails will only be sent if this is defined in the Mascot config file Enter the URL for the local Mascot Server on your LAN A typical
410. ounds Database Editor can be imported using the Compounds Browser window This window is shown Figure 27 4 when the Compounds button is pressed Compounds Browser Calibration x Category Compound General ACTH fraqmenti xv AC XV11 General ACTH _fragmentxeviii_xxxix ACTHsviili_xxHix General Aldolase Ald General Angiotensin_i Angi General Angiotensin_ii Angii General Angiotensin_iii Angiii General Anthranilic_acid Anth General Azothiothymine Att General Bovine_insulin Ins General Bovine_serum_albumin Bsa General Bradykinin Brad General Bradykinin_fragmenti_vii Brad_vii General Caffeic_acid Caff General Carbonic_anhydrase Carb General Cyanohydroxcinnamic_acid Cyano General Cytochrome_c Cytc General Dithranol Dit Show category General compound Y Sort Alphabetic x Mass Average Most Abundant Monoisotopic 2093 4484 C95 H145 N29 0235 Figure 27 4 Compounds Browser window Select a compound from the browser window list and it will be added into the reference list When the entry is complete press the I nsert button to put the new entry into the reference list New entries will be inserted in ascending mass order over writing any entry in the list which is within 1 Da of the new entry mass To delete an entry simply select the entry in the list with the mouse SELECT button the entry will be highlighted when selected and press Delete To clear the whole list and remove all entries press Clear When the l
411. oup is attached to any other site however its attachment will not be prohibited For example if it should only be used to protect Alanine A and Lysine K then type AK into the Modifies entry Introduction 577 578 Chapter 38 Creating a compound database Introduction Defining N and C termini N and C termini for amino acid sequences are also defined in the Compounds Database Select N terminus or C terminus as the Category then click on the New button The Edit Compound window will be displayed with the Terminal Group property page shown as in Figure 38 4 Edit Compounds General Compound Amino Acid Sugar Protecting Group Terminal Group Cation Nucleotide Name Formula Terminus N E I Bi Terminal HPLC Index jo Clear Figure 38 7 Edit Compound window for Terminal groups Type in a Name and Formula for the protecting group The group can have an HPLC I ndex which will be used in calculations of HPLC indices for amino acid sequences Select whether the group defines an N or C Terminus and press OK or Apply Defining Cations Cations associated with amino acid sequences are also defined in the Compounds Database Select Cation as the Category then click on the New button The Edit Compound window will be displayed with the Cation property page shown as in Figure 38 4 Edit Compounds General Compound Amino Acid Sugar Protecting Group Terminal Group Cation Nucleotid
412. ove all Results file destination Eder Dire Filename I Auto select filename Dialog data Load new Save Process Figure 19 7 lon Finder window add entry 3 Click the mouse pointer in the adjacent tolerance cell and type in the required tolerance for that mass Editing an entry Double click the mouse pointer in the required row and make your change s 324 Creating editing a list of masses Chapter 19 lon finder Removing entries Removing an entry 1 Double click the mouse pointer in the required Mass cell You can use the Shift key in conjunction with the mouse pointer to select several adjacent entries Also you can use the Alt key in conjunction with the mouse pointer to select several individual entries 2 Select the Remove entries button mass and its tolerance is removed Removing all entries Select the Remove all button all rows are removed Creating editing a list of masses 325 Chapter 19 lon finder Defining destination of results file You can either specify the filename for the results file or let lon Finder create one for you based on the name of the spectrum Specifying the filename 1 In the lon Finder window select the Folder file button Save As x Save in e mass lists ef Ee Fe mass list txt My Recent Documents E Desktop RA i My Documents My Computer a Sets File name 7 pept
413. oy database search select the Decoy checkbox Mascot PMF searches 299 Chapter 18 Protein peptide analysis using Mascot search engine Mass list Mass list This section allows you to specify the list of masses on which to perform the search You can fetch a range of peaks from the current spectrum To do this enter the bounding masses into the boxes below and then press Fetch peaks The mass list will be updated with the matched peaks Alternatively you can manually enter a set of peaks into the Mass list For search box Lowest peak 700 Highest peak 3500 Fetch Peaks Mass list for search 1 In the Lowest peaks and Highest peak fields enter the required mass range 2 Click the Fetch Peaks button the Select Peaks window is displayed a Set the number of Peaks required the peaks with the highest intensity are chosen automatically b Click the List peaks button the masses identified in your spectrum are displayed Select Peaks BE Dataset 1 sample 12 Automatic Masses 700 3500 Peaks 70 4 Manual Mass 397 5600 ie Edit Mass List Insert Delete gt Selected mass 7 You can add peaks type in the Mass and click I nsert and remove peaks select the required mass and click Delete gt Selected mass 300 Mascot PMF searches Chapter 18 Protein peptide analysis using Mascot search engine 3 The list of masses in the Select Peaks window are
414. pecified and altered by Tuning for an acquisition double clicking on the precursor ion list in the laser firing window see Figure 14 5 on page 142 Resolution This controls how tightly the precursor ion selection will be Normally the 250 resolution window would be recommended as this will generally retain a complete isotopic distribution but will reject other close distributions CID control Specifies the amplitude of the excitation waveform that is used in fragmenting the precursor ions A value of around 300 is recommended as a good starting point However some ions fragment more easily than others and it may be necessary to vary this parameter to obtain the best quality fragmentation NB If this value is set to 0 then the correct precursor selection may be verified Chapter 14 Collecting data from a sample Chapter 14 Collecting data from a sample 139 140 Chapter 14 Collecting data from a sample Introduction Data collection is controlled via the laser Firing tab of the Acquisition dialogue Figure 14 4 and Figure 14 5 initiated from the Instrument Acquisition option on the base window see Figure 14 1 below This tab allows for the selection of samples for analysis aim positions on the sample spots laser power number of profiles and other settings which are related to laser firing 4 angio2_msms0006 p14r_msms0004 MALDI MS Biel x File Edit Yiew Instrument Automation Processing Help
415. play Spectrum v Profiles 1 Masses 12 1040 1 gt Saye GUS Save As Open Auto Experiment Results Comments Parameters Print Ctrl P PrinbReporn Print Preview Print Setup Export Exit Comments a x Current comments E Dataset 1 bill angio 2 with theoretical0002 Title Prefix Select well l List fan entries z Figure 10 1 The Comments window Introduction Chapter 10 Putting comments with collected data When the data is reviewed at a later date any relevant information is readily available on the samples and conditions under which the data was collected The Comments window has numbered sections into which any type of information can be entered A single line per sample can be used for a ten twenty or thirty sample slide or multiple comment lines can be used for a continuous slide The comments are shown on a scrolling list window Sample comments are automatically displayed above the spectrum ioi File Edit View Instrument Automation AE HEC 2 8 Aare Display Spectrum Profiles 1 54 Masses 1046 1060 KEA else 2 ms ms fragmentation Calibration well using PT4R Copy comments from gt z Clear comments Load file Save file 1046 For Help press F1 Apply to Current dataset x OK Apply Cancel 1048 1050 1052 1054 1056 1058 1060 Mass Charg
416. ple This dataset name will be inserted in the first entry slot on the Load data window Any previously loaded dataset in slot 1 will be automatically unloaded when data collection starts For a fuller explanation of run names and datasets refer to Loading data on page 75 mode checks When the Fire button is pressed the software will always check to see whether the instrument is fully pumped down and will initiate the required pumping to achieve a satisfactory vacuum before data collection can begin If the sample stage is out it will be retracted and the door closed Laser firing can only begin if the Experimental technique tab dialogue Mode option is set to Operate A message will be reported if this is not the case simply switch to Operate and the firing sequence will be resumed When no laser shots have been fired for 1 hour the instrument will automatically be put into standby mode Data space checks If data storage has been selected the computer will estimate the amount of hard disc space required in which to store the data This estimate is based upon the number of profiles being stored and the number of profiles being averaged together The amount of data actually collected can be more or less than the computer s estimate A warning message will be displayed if the computer estimates that there is insufficient space on the hard disc for the amount of data which it expects to be collected Under these circumstances
417. ples A group of samples consists of a selection of plate wells and an associated method created using the Method Editor This provides the ability to process samples in different ways depending on the user requirements and to associate samples that have similar analysis requirements together Auto Experiment 203 204 Chapter 15 Automated operation There are three control groups within the Auto Experiment window each one having a particular scope of functionality All the buttons in toolbars are of a type that will display a tooltip which is a brief description of the purpose of the button and appear raised when the mouse pointer is stationary over a button Some buttons are automatically disabled depending on the current state of the experiment All button functions are activated by a left mouse button click Plate Controls The plate controls are used to select the type of plate used in an experiment select and de select samples using the mouse pointer and zoom in and out to display more or less of the sample plate There are four controls in the group including one toolbar the overall view of all the samples selected for the current experiment the current group of samples selected the detail of current group of samples and the plate toolbar There is a text box that is used to display the description of the currently selected plate this is located directly above the toolbar Wells are selected by either a single mo
418. pound Defining amino acids The Compounds Database is shipped with 23 amino acids definitions already entered These can be used in either the abbreviated form e g Arg Asn Asp or the short form R N D within compound formula definitions If using the abbreviated form of notation for the amino acids then the three letter mnemonics can be used Table 38 1 gives some examples Table 38 1 Short symbol amino acid formulae Formula Meaning Tyr Trp Ser Tyrosine Tryptophan Serine Tyr2 Trp4 Ser9 2xTyrosine 4xTryptophan 9xSerine Chapter 38 Creating a compound database Table 38 1 Short symbol amino acid formulae Formula Meaning Tyr2 Trp4 2xTyrosine 4xTryptophan Ser9 5 9xSerine x5 The rules for setting the number of each amino acid and grouping using parenthesis are exactly the same as for periodic table elements When using the single letter short form amino acid notation brackets should be placed around any sequence of amino acids to avoid confusion with individual elemental formulae e g where W could be Tryptophan or Tungsten as shown in Table 38 2 Table 38 2 Single letter amino acid formulae Formula Meaning YW S Tyrosine Tryptophan Serine Y2 W4 S9 2xTyrosine 4xTryptophan 9xSerine Y2 W4 S9 5 2xTyrosine 4xTryptophan 9xSerine x5 Amino acids may be defined within the Compound Database by selecting Amino Acid as the Category and clicking on the New button The Edit Compound window will
419. principal benefit of enzymatic or chemical cleavage is that larger proteins are broken down into smaller more manageable units These digestion products can be monitored by MALDI techniques profiles collected from the peptide sample can be taken at varying stages of enzyme digestion and the cleavage products monitored The Digest fragments section controls the contents of the enzyme report section A separate report section is generated for each of the digest products A typical report is shown in Figure 42 22 The and buttons on the Display Toolbar can be used to step to the next and previous page The Sal and A buttons resize the text p14r_msms0004 MALDI MS ojx File Edit View Instrument Automation Processing Help fia Aee 2 Sl e E Dispense CacusterFiesuts z Potes 1 227 gt Masses 18032500 I I Untitled L 9 YGGFLRRIR trrryprrrey 10 N terminus Hydrogen H C terminus Hydroxy H O Sequence composition F G2 I L R3 Y Elemental composition M H C52 H85 N18 011 Average mass M H 1138 37 Bull Breese 2360 00 Most abundant mass M H 1138 13 HPLC 38 50 Monoisotopic mass M H 1137 66 Trypsin digest Average Masses M H N Position Mol Wt BB HPLC Elemental Formula Sequence 1 1 6 712 83 2290 0 45 7 C34 H50 N9 08 YGGFLR 2 Toe T 175 21 690 0 3 0 C6 H15 N4 02 R 3 9 288 37 760 0 9 6 C12 H26 N5 03 IR For Help press F1 CAP NUM YW Figure 42 22 Example of a report for enzyme
420. processed data set this can be set in the Spectrum Display Contents window 4 Press the Combined Cal button shown below in Figure 27 16 Calibration Auto calibrate Fit through zero I Correct Load Save Tolerance fo SJ a gt tl T Calibrate Combined Cal Figure 27 16 Calibration window options for combining calibrations Calibration of the instrument Chapter 27 Instrument Calibration This will start up the Combined calibration window shown in Figure 27 17 below Data to combine for calibration 1 pl4r_msms0004 I 2 angio2_msmsO006 Figure 27 17 Combined calibration window The window shows all of the currently loaded data sets 5 Inthe Combined calibration window select only the data sets which are to be combined as a single calibration 6 Ensure that a list of suitable calibrant references have been entered in the main Calibration window 7 Press the Calibrate button in the Combined calibration window 8 Save the calibration as described in Saving calibrations on page 474 This technique allows a calibration to be built up over a large mass range using calibrant compounds which for a number of reasons may be impossible to prepare as a single mixture Calibration graphs A graph showing the difference between the reference mass positions and the actual masses calculated from a least squares fit line through all of the reference points is available This graph g
421. profile scale on the diagonal axis This is done by setting the parameter Front to Mass This produces a chromatogram display with the mass m z axis at the front as in Figure 17 12 showing how the mass spectrum changes from profile to profile Displaying Chromatograms Chapter 17 Viewing the collected data olnt 100 11 mY mass 1000 to 15000 Smoothed 1 ee Sl TFS ES an LEE ZZ eres SS SF DA P A RA RRA AAAA LEB FS LLL EE RE LLLI LIEK ILILILILIL ILII fo LEA LLL ALT LEERE RRRA RRR eA A A A e A e A A ooo A A A AA 5000 10000 15000 Mass Charge Figure 17 12 Rotated 3D contour map of collected data Expanding 3D chromatogram displays Regions within these displays can be expanded in the same way as with spectra Either the new mass range and range of profiles can be typed in to the respective entries on the window or the mouse can be used to select the new range In the case of 3D chromatograms the mouse provides a flexible method of expanding either the range of profiles mass range or both profile and mass ranges simultaneously Selecting a range of profiles on a 3D chromatogram Using the mouse move the mouse pointer to a position on the graph at the start of the required range of profiles Press and hold down the mouse SELECT button and drag the mouse horizontally along the range of profiles to the required end point of the range To force the mouse to move in one direction only hold the
422. ptide sequencing work the task at hand may be easier since the compound may be the product of an enzyme digest where cleavage points along the protein peptide chain are well known In all cases a list of possible constituent species is required This Chapter 39 Searching for molecular weight matches is entered in the top of the window as a list where each species is specified along with a specific number or range of numbers of the species present In the example shown in Figure 39 2 there may be up to twenty occurrences of the amino acids Glycine Proline and Serine The amino acids which may be present are entered in the search list Any element compound or species defined in the Compounds Database window can be entered in the Search window list Next the observed mass difference or fragment mass the molecular weight for which a match is sought is entered in the middle section Mass entry A Tolerance window is needed to restrict matches to a specified mass window about the required molecular weight The tolerance can be in Da mDa ppt or ppm Search BEI Help Fomula oue Pro 0 20 Ser 0 20 Trp 0 20 Val 0 20 Formula val Quantity 10 20 Insert Delete Clear list Mass 1000 Da Tolerance 2 a Da he Isotopes Average g Match 1 of 20 Mass 1000 0324 Daltons Error 0 0324 Daltons 32 39 ppm Formula Quantity Figure 39 2 Search window ecoceoe uw un 586 Chapter
423. quence s will be fragmented according to the selections made on the Fragmentation window The fragment masses generated will then be compared with the peaks selected in the Select Peaks window Where a match occurs a report will be generated showing the fragments found and the mass difference for all of the peaks A Stop button is provided to abandon very long matching operations Finally any or all of the match results in the Multiple sequence results section can be sent to the printer by using single clicks of the SELECT button to make the selections from the list then press the Print selections button Introduction 639 640 Chapter 42 Sequence Calculator An example of a peak match report is shown in Figure 42 33 Trypsin digest fragment number 1 I Plasminostreptin PSTI type proteinase inhibitor Streptomyces GAGGDFDALTVR UE PCE Ey Ee EZET 10 20 N terminus Hydrogen H C terminus Hydroxy H 0 Sequence composition A2 D2 F G3 LRT V Elemental composition C50 H80 N15 018 M H Average mass 1179 2783 Bull Breese 1930 00 M H Most abundant mass 1178 5806 HPLC 51 70 Fragments Average Masses M H Fragment Predicted Actual Difference Digest 1179 2783 1A 30 0497 30 9000 0 8503 2A 101 1287 101 1300 0 0013 3A 158 1807 9 4A 215 2327 215 2200 0 0127 SA 330 3214 6A 477 4986 477 5500 0 0514 7A 592 5872 BA 663 6662
424. quences The Load sequence window Figure 42 9 is used to load a sequence from the database into the viewing panel Select Load from the File menu amp Sequence Calculator Oy x File Edit View Settings Save Load Sequence x Database Shimadzu Biotech Launchpad Peptides angiotensin 2ksq Select Clear Keywords Sequence Import Mass range 1 1700 daltons List 10 entries Delete Keywords Mass Sequence name U o0 1047 20 Angiotensin 2 4 H Listed 1 out of 1 matches Figure 42 9 Load Sequence window Introduction Chapter 42 Sequence Calculator The Load Sequence window allows the user to search through any of the peptide databases which are present in the path defined by the Configuration Editor as the Peptides folder on the computer network see Environment Configuration Editor on page 60 The Sequence Calculator assumes that all peptide databases are located in this folder You may specify any location on the network using the Configuration Editor As mentioned at present two commonly occurring commercial database formats are supported the NIH database format fasta and the EMBL database format seq Any sequences created within the Sequence Calculator are stored in a proprietary database format ksq The reason for this is that as yet a standard format is not available to define cross linked sequences The Load Sequence window al
425. r 2005 13 47 Cal tof 4 Mar 2005 10 10 CID Shimadzu Biotech Kompact MALDI 6 v2 7 0 Build 20050623 Mode reflectron_ms_ms Power 74 G lnt 28 mV sum 6223 mV Profiles 25 245 Smooth Av 10 1404 100 90 80 70 60 50 40 30 20 i 1272 32 10 0 800 1000 1200 1400 Mass Charge Figure 36 2 An example of a Print Preview 561 Chapter 36 Printing the contents of displays 562 Chapter 37 Element database Chapter 37 Element database 563 Chapter 37 Element database Accessing the database All of the calculations within the MALDI MS suite of software which involve formulae or elemental masses refer to a database of elemental isotopic abundances This database can be viewed by using the Elements window To start the Element Database select Element Database from the MALDI MS programs menu on the Taskbar Figure 37 1 Launchpad SEI Show on startup V _Heb Click the Element database icon B Ja cl aia MALDI MS Search Archiver Enzymes SaLe Or use the Start menu system Femen EOoU dS ERr Programs IT Documentation e Ey H Archiver w Favorites gt a 7 i Compounds Database 5 ccessories 2 Documents Configure Environment ao a Administrative Tools E vi Settings Mi Shimadzu Biotech Launchpad PET Element Database Editor 3 a Search a ay e Enzymes 9 eare an RoboHelp Office 2 Launchpad g 2 Help and Support SmartDraw 2
426. r are accepted and swapped over if necessary All i e negative and i e positive peaks within are then flagged before specific mass filters are flagged Only masses or chemical formulae and any preceding symbols lt gt are interpreted The symbol may be separated from the mass or formula by white space If the algorithm is unable to parse a particular line it is ignored and the next line is attempted Filtering specified peaks 263 264 Chapter 16 Cleaning up data Filtering specified peaks Chapter 17 Viewing the collected data Chapter 7 Viewing the collected data 265 266 Chapter 17 Viewing the collected data Introduction The displays in the MALDI MS window Figure 17 1 offer one of the most comprehensive and flexible systems for processing and viewing collected data Any number of simultaneous displays can be created with the ability to have real time updates in selected displays as data is collected Insets displays within displays can be created providing enlargements or comparison views of data Displays can be enlarged reduced made to be the full size of the window or arranged to suit almost any requirement Most importantly the results displayed in the base window can be exported in standard Windows formats so that the results can be used in desktop publishing applications for producing data reports which can be easily edited for presentation or publication if angio2_m
427. r checking file and directory write permissions on the databases directory being written to Also check disk space Error 18070 Error writing compound unknown group A compound was being written out from the database but the group was unknown please check the Compounds database program Error 18080 Insufficient memory Close other programs shutdown active processes to free more memory Error messages 655 656 Chapter 44 Summary of error messages Error 18090 Formula too complex formula Error 18100 Bracket expected at symbol in formula formula Error 18110 Error at symbol in formula formula Error 18120 Symbol not known symbol in formula Error 18130 Incorrect formula formula The above errors indicate that symbols were found in the formula which were either invalid symbols or the formula was incorrectly written syntax error See Rules for entering formulae on page 573 Error 18140 Memory allocation failed returning formula unaltered Close other programs shutdown active processes to free more memory Error 18150 Formula to elements conversion failed returning formula unaltered Symbols were found in the formula which were either invalid symbols or the formula was incorrectly written syntax error See Rules for entering formulae on page 573 Error 18160 The isotope value entered is not in the elemental database Error 19000 Unable to load the Dzip32 dll Check that this file exi
428. rage mass values to be used in the experiment Overview Select to show the overview table in the final results tile The overview table is more suited to the MS MS results tile Report top hits Specify the maximum number of hits to report in the results tile Mass Values Parent search only Specify the peptide mass value in a parent search as either the charge carrying proton MH neutral Mr or negative M H MS MS tolerance MS MS search only Specify the error window around the fragment ion mass Select units between Da or mDa Peptide Charge MS MS search only Specify the peptide charge state in a MS MS search For Axima Confidence the precursor peptides will generally be MHt hence the peptide charge should generally be set to 1 Method Editor 181 182 Chapter 15 Automated operation ICAT MS MS search only Select to perform the CAT lsotope Coded Affinity Tag method Selecting this limits the search to cysteine containing peptides also adding heavy and light ICAT tags to the variable modifications Instrument MS MS search only Specify the description which best describes the Instrument used to acquire data This setting determines which fragment ion series will be used for scoring The Mascot interface is designed based on parameters of the most recent version of Mascot currently 1 9 at time of press Some parameters may be obsolete if running older versions of Mascot For a more
429. ram and their possible states are described in the table below 98 Table 11 1 Axima Confidence status diagram key Item Status Explanation Laser Off On The laser is shown in the Off colour blue when not ready to fire and in the On colour green when ready Rotary Always on This pump is used to pump the pump no inlet chamber from atmosphere indication and to pump the exhaust from colours the turbo pumps Turbo pump Fail Off The pumps provide the high Accelerating vacuum They are only or At speed switched off when the instrument is vented The turbo pumps are shown accelerating when the blades are running at less that 80 of full speed Vacuum Fail Poor The gauge is always on The gauge OK gauge reads pressure in Millibar Pascal and or Torr units via a context menu over the window HT supplies Fail Off On The supplies are switched on when the instrument is fully pumped and enabled see Preparation for data collection on page 117 They are always switched off before the door is opened V1 Open This valve isolates turbo pump SAC backing Closed 1 during analyser pumping valve V2 Open This valve isolates turbo pump Flight tube Closed 2 during SAC pumping backing valve Axima Confidence instrument status Chapter 11 Checking the instrument status Table 11 1 Axima Confidence status diagram key Continued Item V3 SAC turbo vent valve V4 Flight tube turbo vent valve
430. raphs such as a Spectrum plot may be copied on to the clipboard as bitmap images and pasted into for example a word processor application The clipboard operations are available from the base window Edit menu J p14r_msms0004 MALDI MS Oy x File Edit View Instrument Automation Processing Help lie Undo Copy Paste Delete Copy Window Copy Selected Tile Copy Entire Report Ctrl Z P ind Display Mass List Profiles 1 v Masses 12 1040 I gt i Ctrl C Ctrl Del Figure 35 1 Clipboard functions on the Edit menu Text in for example a Mass list report in the current tile may be copied by highlighting it in the usual manner then selecting Copy from the Edit menu A complete text report in the current tile may be copied to the clipboard by selecting Copy entire report from the Edit menu It is now available on the clipboard for pasting into a word processor To copy a spectrum as a bitmap image onto the clipboard make the spectrum tile the current one then select Copy selected tile from the Edit menu Alternatively all of the tiles on display may be copied to the clipboard by selecting Copy window from the Edit menu Note that there are also facilities for exporting an entire window or a selected tile as an enhanced metafile see Exporting data displays as meta files on page 530 Chapter 36 Printing the contents of displays Chapter 36 Printing the contents of displays 559 560
431. rch template Settings Add entry 757 0000 2 0000 1046 0000 1 0000 Paste data Import data xj Remove entries Remove all 1297 0000 1 0000 Results file destination Folder C Documents and Settings Adr _ Folderifile _ Filename IV Auto select filename Dialog data Load new Save Figure 19 11 Auto select filename Existing filenames If the filename within the lon Finder window already exists lon Finder will append the date and time to the filename to avoid overwriting the existing file gion Finder BBE m Ion search template Settings Add entry Paste data Import data A Remove entries Remove all Results file destination Eolder C Documents and Settings Adr Folder file Filename 7 peptide mix date070208_091 Auto select filename Dialogdaa Load new Save Figure 19 12 Filename appended 328 Defining destination of results file Chapter 19 lon finder Saving and loading lon Finder settings This feature allows you to save the current settings so that you can retrieve them at a later date The following is saved to a file Mass and Tolerance data All the settings within the lon Finder settings window Folder and filename information Saving your settings 1 In the lon Finder window select the Save button Save As 1 1 x Save in e mass lists ct EE Fe m
432. re based on a delimited text file such as is commonly exported from a spreadsheet program One line of text contains all the information required to describe a single sample Each line is divided into fields separated by a single character that is only used for field separating this must be a space or tab e Lines are terminated with a carriage return or line feed character e Any lines encountered during file import which do not adhere to the expected file format are rejected The order of fields is fixed though not all are required ASCII text experiment file formats Chapter 15 Automated operation The following table describes the fields Table 15 9 Delimited Ascii text Experiment file fields Field Name Well ID Sample ID Data File Obligatory YES YES NO Description The well identifier e g A1 To specify an irregular well pattern such as might be expected with a 2D gel specify spot location and size in the format Welll D x pos y pos diameter where x pos Horizontal distance mm from plate left hand edge to spot centre y pos Vertical distance mm from plate bottom edge to spot centre Figure 13 8 on page 125 diameter spot diameter mm N B Parentheses and commas must be supplied there must be no space between WelllD and the openening parenthesis The identifier for the sample this will be placed into the comment field for the sample If this field req
433. reflected in the Mass list for search field Mass list This section allows you to specify the list of masses on which to perform the search You can fetch a range of peaks from the current spectrum To do this enter the bounding masses into the boxes below and then press Fetch peaks The mass list will be updated with the matched peaks Alternatively you can manually enter a set of peaks into the Mass list For search box Lowest peak 700 Highest peak 3500 Fetch Peaks Mass list for search 4 You can also amend the mass list in the Mass list for search field Searching the Mascot database Cancel 1 Click the Search button the Database search window is displayed while your PC connects to the Mascot search engine Database search xi A search is currently being performed The progress of the search can be seen below Search status Communicating with the search engine If there is problem accessing the search engine details are provided within this window Mascot PMF searches 301 302 Chapter 18 Protein peptide analysis using Mascot search engine 2 When the search is completed the results are displayed in your web browser for example Internet Explorer MATRIX j baerce Mascot Search Results User Your name Email your name kratos co uk Search title Arabidopsis thaliana MS experiment Database NCBInr 2464940 sequences 834614836 residues Taxonomy Arabidopsi
434. rent colour to distinguish them from other peaks in the spectrum Producing a table of peaks in a spectrum 279 Chapter 17 Viewing the collected data Tick the Significant peaks only box if only peaks which have been flagged as significant are to be reported This is especially useful in polymer analysis to find the percentage of the total area of the detected polymer series represented by each peak The page up and page down display toolbar buttons are used to move page by page through a text report See Table 20 3 on page 340 for a summary of the text report navigation controls on the display toolbar For information on printing text reports see Printing the contents of displays on page 559 280 Producing a table of peaks in a spectrum Chapter 17 Viewing the collected data Displaying Chromatograms Chromatograms show the variation of intensity with each profile and can be particularly useful in locating the sweet spot ona sample The sweet spot is simply the area of the sample spot which produces the most ions This is usually caused by crystallisation creating pockets of increased concentration of the sample in certain areas of the sample slide Set Display to Chromatogram The chromatogram shows one intensity value for each profile The intensity can either be the average intensity of all data readings in the profile or the largest intensity in the profile over the mass range entered The sample number ra
435. rint Print the Instrument conditions instrument name Method Editor 95 Chapter 15 Automated operation Table 15 3 Spectra Properties Continued Print Print the title from the comments window comments title Print folder Print the folder where the data is stored as name well as the dataset name Print borders Print borders around the current display Print 1st Print the first comment from the comment comments window Table 15 4 Sequence Report Properties Select whether the section heading of the Print section report is printed The heading appears at headings the top of each page above any column headings Print blank Prints a blank line after the section heading line after section headings Print column Print column headings headings Print blank Prints a blank line after the column line after headings column headings Defines number of lines to print on each see to prin page of the report Table 15 5 Mass List Contents Settings Set the decimal placing of the listed masses Precision Maximum Specify the number of most intense peaks listed peaks in the mass range to be printed 196 Method Editor Chapter 15 Automated operation Table 15 5 Mass List Contents Settings Set the minimum peak apex millivolts value Peaks below this height are not printed Minimum peak apex Significant Print peaks which have been flagged as peaks only significant only The Window properties has no settings as the curr
436. rming up At low resolutions this will have no effect but at high resolutions you may miss required ions If this drift affects your acquisition leave the Axima in operate mode and fire the laser on to a well with no samples for approximately one hour After this warming up period the ion gate will filter the required ions Acquiring MS MSn data on Resonance 63 Chapter 14 Collecting data from a sample Acquiring MS MSn data on Resonance Chapter 15 Automated operation Chapter I5 Automated operation 165 Chapter 15 Automated operation Introduction With the introduction of the Axima range of instruments a much greater degree of automated operation can be utilised The two options to setup and start automated instrument control are found on the Instrument menu Figure 15 1 shows how to start the first of these windows olx angio2_msms0006 p14r_msms0004 MALDI MS F Edit View Instrument Automation Processing Help BEA He amen a Protes gt Masses i2 1040 fe Auto Experiment LC MALDI iTRAQ Method Editor default mtd ChIP _ ChIP Imaging Ext Ext m Sample Method E 3 Sample Method default mtd a ME Parent H WEY Acquisition MRA Raster Mx Auto Quality v t Laser Firing El WEY Processing ML Calibration EZ Peak Clean MIME Monoisotopi MEA Peak Filterin MS Mascot Pare a r Setup Configuration default_reflectron Mass Ra
437. rring In this Summary a word is shown in square brackets when the error message given may show a specific value e g time name or number Some faults can be cleared by switching the Axima off and then on using either a switch at the mains supply or the on off switch at the back of the instrument Error O Error 10 Error 1000 Error 1030 Error 2000 Error 2010 Error 3000 Error 4000 Error 4000 Error 4010 Chapter 44 Summary of error messages Error messages An unregistered error has occurred See log window for more information Insufficient memory is available Close other programs shutdown active processes to free more memory An Error occurred whilst storing data The Windows event log window may contain more specific descriptions of the errors encountered check file permissions on the directory being written to Cannot open sample door This message appears if the vacuum controller is not ready to open the sample door at the end of a laser firing sequence or when changing slides It may be due to use of the manual door controls during data collection An Error occurred while writing the file Do you have permissions for this directory or is the disk full Check file and directory write permissions on the directory being written to also check whether the disk is full An error occurred while reading the file Is this a MALD MS labels file Is this file a valid labels file t
438. rt is composed of Section headings only so ensure that the Section headings option on the Text Report tab of the Display Options window is selected see Figure 20 35 on page 380 otherwise the report will be comprised of blank pages Figure 42 24 on page 627 gives an example of the enzymatic digest and A 17 and B 17 fragment ions produced from the MALDI analysis of Dynorphin I Dynorphin trypsin digest fragment number 1 L 6 H C terminus Hydroxy F G2 ILR Y C52 H85 N18 011 1138 37 Bull Breese 2360 00 1138 13 HPLC 38 50 1137 66 HO N terminus Hydrogen H C terminus Hydroxy Sequence composition F G2 LR Y Elemental composition M H C34 H50 N9 08 Average mass M H 712 83 Bull Breese 2290 00 Most abundant mass M H 712 67 HPLC 39 10 Monoisotopic mass M H 712 38 HO Singly charged Fragments Average BB HPLC Elemental Formula Sequence n e 17 b 17 2290 0 45 7 C34 HSO N9 08 YGGFLR 690 0 3 0 C6 H15 N4 02 R 119 147 18 760 0 9 6 C12 H26 NS 03 IR 176 204 24 261 29 408 47 521 Masses M H I Dynorphin trypsin digest fragment number 2 L 1 N terminus Hydrogen Sequence composition Elemental composition Average mass M H Most abundant mass M H M H IMonoisotopic mass Singly charged Fragments Average Masses M H n a 17 o een 1 112 19 b 17 140 20 Introduction H C terminus Hydroxy HO N terminus Hydrogen R Sequence composit
439. rticular order but they must follow any other fields etext C data foldera fileAl C methods meth1 mtd i Method Tab white i space Data File Sample ID Figure 15 33 ASCII Experiment file example input for a predefined plate ASCII text experiment file formats Chapter 15 Automated operation Next is a file suitable for a free hand plate where sample spot location and size is specified In the examples shown A1 71 98 5 First sample text C data foldera fileAl C methods mett A2 62 88 4 2nd sample text C data foldera fileA2 C methods metl B1 44 60 2 Third sample text C data folderb fileB B2 22 7 2 4th sample text C data folderb fileB The centre of Sample B2 is located 22 mm horizontally from the left hand edge of the plate and 7 mm vertically from the bottom edge of the plate the sample is 2 mm in diameter Note the use of commas to separate the numeric location and size entries Also note that there is no space between B2 and in the Well ID field Figure 15 34 ASCII Experiment file 2D gel example In either case Al and A2 will be acquired into two different files file Al and file A2 both in folder of C data Whereas B1 and B2 are both acquired into a single file fileB in folderb of C data Samples B1 and B2 will use method file meth1 mtd because it is the last method specified and as such supplied as default to samples B1 and B2 which have no method specified ASCII text experiment file formats
440. s Cannot create output directory The specified directory is not valid Cannot create output directory Access denied Cannot create output directory The parameter file does not exist Too many parameter files specified Invalid parameter file specification Chapter 34 Batch processor XML export You have misspelled the mzXML contents specifier or entered a content type which does not exist For example mzxml foo Alternatively the user has attempted to set output contents for mzData This error will appear if you specified both mzxml and or any derivatives and mzdata You have specified a source folder which cannot be found This error may occur if you omit quotes from a path which includes spaces You have specified more than one source folder You have specified more than one destination folder You have incorrectly specified an output directory using invalid characters Incorrect permissions or sharing violations made the directory unable to be created The output directory was not able to be created The exact reason for this error could not be established You have specified a processing parameter file which does not exist This error may occur if you omit quotes from files with spaces in the path You have specified more than one possible source of processing parameters for example by specifying p d and p c The parameter specification should be p d or p c If yo
441. s Desktop 98 00 09 51 98 00 09 51 98 00 09 51 100 00 10 100 00 10 c RAY 101 00 10 101 00 10 c My Documents 102 00 10 1 95 00 09 33 1134 000 102 00 10 1 95 00 09 33 1134 Imz0001 run 102 00 10 1 96 00 09 39 1061 0001 run 102 00 10 1 96 00 09 39 1061 Imz0001 run 107 00 10 4 um gt l A Fie name 34 00 09 27 898 m20001 run 75 00 07 291 Places i Files of type MALDI Data Files run z Cancel A My Computer Figure 34 4 Open window showing run files 4 Select the Open button to add the files to the Batch Processor IES Batch Processor Processing Type mzData Converter x OPOS Processing Settings Source Files File B C Documents and Settings bill KRATOS My Documents iTRAQ Results iTRAQ 2 1 117 114 75 00 07 29 1109 0001 run Q C Documents and Settings bill KRATOS My Documents iTRAQ Results iTRAQ 2 1 117 114 75 00 07 29 1109 Imz0001 run P C Documents and Settings bill KRATOS My Documents iTRAQ Results iTRAQ 2 1 117 114 76 00 07 35 1245 0001 run C Documents and Settings bill KRATOS My Documents iTRAQ Results iTRAQ 2 1 117 114 76 00 07 35 1245 Imz0001 run P C Documents and Settings bill KRATOS My Documents iTRAQ Results iTRAQ 2 1 117 114 79 00 07 57 1636 0001 run P C Documents and Settings bill KRATOS My Documents iTRAQ Results iTRAQ 2 1 117 114 79 00 07 57 1636 Imz0001 run P C Documents and Settings
442. s Opening and saving parameter sets Parameter sets are opened and saved using the File menu Parameters option Figure 9 1 angio2_msms0006 pi4r_msms0004 MALDI MS 0 Eg File Edit View Instrument Automation Processing Help Open Ctri O Display Spectrum gt Profiles fi Masses 12 1040 Ka Save Goes Ki Save As Open Auto Experiment Results Comments Parameters Open Print Ctrl P Print Report Save Defaults Print Preview Print Setup Export Exit Figure 9 1 Parameters menu To open a named parameter set select Open from the menu option This will display a window from which the parameters file to be opened can be selected Figure 9 2 Select parameter file 21x Save in fo Parameters gt ef Ee My Recent Documents E Desktop 9 My Documents PE My Computer a e UM ETna File name three pane layout z Places Save as type Parameter files tofparams x Cancel Opening and saving parameter sets 8I 82 Chapter 9 Parameter sets Figure 9 2 Select Parameter File window This will set all of the instrument control and data processing settings to the values stored in the named parameter set All windows which were displayed when the parameter set was saved will be shown in their original positions The current settings on all of the windows can be saved to a named parameter set by selecting the Save optio
443. s To label all displayed datasets set Label to All To label only specific datasets use Selected and highlight the names of the datasets to be labelled in the list To switch automatic labelling off set Label to None The centroided and processed peaks can be labelled with mass labels above the centroided or apex mass positions Labels are positioned just above the peaks with a small tic mark indicating the position of the centroid or apex of the peak Labelling is performed in such a way as to avoid overlapping or clashing labels generating a clear uncluttered report Three types of mass labels are available Mass Difference or Relative To display mass labels on the peaks set the Label type option to Mass The Mass labels option is used to specify the number of decimal places shown in the labels this can be either 1 decimal or 2 decimals Chapter 25 Automatic graph labelling scaling and printing Labels are displayed on both the processed and peaks graphs of a spectrum distribution or reference display This allows the processed data to be compared with the centroided peaks found in the collected data Labels can be set to appear above a certain intensity threshold this is done with the above option Set the percentage intensity threshold above which labels should be displayed This allows small insignificant peaks to be ignored while peaks above a specified intensity will be labelled To label all peaks set this value
444. s table Introduction 595 Chapter 40 Polymer simulation 596 Introduction Chapter 41 Defining Enzymes Chapter 41 Defining Enzymes 597 Chapter 41 Defining Enzymes Enzymes used in peptide digests can be defined using the Enzyme Database program Their cleavage sites and mode of digest can be defined by a set of rules These rules are then used in the sequence calculator to determine digest products To start the Enzyme Database window select Enzyme Database from the MALDI MS programs menu on the Taskbar Figure 41 1 Launchpad 1 x Click the Enzymes icon MALDI MS Search Archiver Enzymes Daae Or use the Start menu system oe eee Pr ograms Documentation fA archiver 3 Compounds Database E Favorites an Accessories 2 Configure Environment A Documents PER 5 i me Administrative Tools t Element Z os Settings gee Shimadzu Biotech Launchpad fa Element Database Editor 2 Q aa Quadralay gt Search i f o t RoboHelp Office gt fenced Help and Support See e Log Window a P SoundMAX aoe B MALDI MS pea J Run I Startup i D a P i x rs Polymers ymantec Client Security 5 eee cea X Sample Plate Editor j m Volo View Express p se p g Search z Turn Off Computer Figure 41 1 Starting the Enzyme Database program The Enzyme Database window wi
445. s Average masses Md ERK Peak Cleanup rE z ZIM Monoisotopic Peak Picker oe as MEX Peak Filtering VS Mascot 5 5 E Output en Load Save Filters from Peaks gt Data Storage Get Manual Settings Save Method Load Method Figure 15 14 Method Editor Peak Filtering Parameters for the peak filtering window can be setup as detailed in Filtering specified peaks on page 259 Mascot Searching The Method Editor Mascot window provides an interface for defining protein search parameters which are used to submit mass spectral data to the Mascot database search engine The Mascot Search engine uses this mass spectral data to identify proteins from primary sequence databases There are two versions of Mascot in the Method Editor a Parent Mascot search 178 Method Editor Chapter 15 Automated operation and a PSD Mascot search Similarly the Resonance QIT version contains both an MS and MS MS Mascot search screen Before performing a Mascot search the Mascot Setup parameters must be defined as detailed in Mascot Setup on page 62 To access the Mascot windows select the Mascot label from the tree as shown in Figure 15 15 and Figure 15 16 below Method Editor default mtd BBE Sample Method Sample Method default mtd Mascot Parent Search iy Parent WEY Acquisition SeachTite v l Raster VIX Auto Quality Database MSDE 7 Decoy L Vi 3 Laser Firing D E Processing Report Top Hits 2
446. s the start and end points of the detected peaks the peak limits can be marked on the graph Ticking the Peak limits marker option on the Graphs property page causes markers to be drawn on the processed trace indicating the start and end samples of each peak detected in the data as shown in Figure 25 4 446 gt Markers displayed on a spectrum Chapter 25 Automatic graph labelling scaling and printing lnt 100 6 mV sum 637 mV Profiles 0 100 Smooth Av 10 Baseline 40 5686 0 100 80 60 40 204 5650 5700 5750 Mass Charge 5600 Figure 25 4 Example of peak limits markers Markers displayed on a spectrum 447 Chapter 25 Automatic graph labelling scaling and printing Marking the threshold baseline Depending on which Threshold has been selected on the Processing window the level of the baseline which has been subtracted from the total signal can be displayed on the averaged graph by ticking the Threshold marker option on the Graphs property page This gives a clear indication of the extent of the signal being removed from the data Figure 25 5 Threshold 100 80 60 40 20 5383 2 3157 5 3254 2 100 80 60 40 20 Threshold Figure 25 5 Examples of threshold markers 448 Markers displayed on a spectrum Chapter 25 Automatic graph labelling scaling and printing Labelling peaks Peak markers Peak markers allow the mass positions of expect
447. s 1 54 Smooth Av 20 1046 5 Processed 100 1047 5 a 1048 5 o Yolnt 7148 mY Profiles 1 54 Threshold 25 Centropaaks centroid apex 1046 5 100 50 1048 5 1049 6 1046 1048 1050 1052 1054 Figure 17 3 Four types of traces available for spectra In the Spectrum contents window click the mouse SELECT button on a trace button to add or remove that trace from the report display Up to ten loaded datasets can be displayed simultaneously in a single display each trace will be displayed in a different colour to allow individual traces to be distinguished The colours of the traces can be set using the Spectrum colour editor available from the Display Options window Graphs tab see Choosing user defined colour schemes on page 429 Choosing the sample to display If you are about to collect data it is not necessary to set the displayed sample spot number or the range of profiles on the base window as these are set automatically The Spectrum or Chromatogram displays will show data as it is being collected and the traces will be updated accordingly Displaying Spectra Chapter 17 Viewing the collected data However after data collection has stopped the data for a particular sample spot on the slide can be displayed by typing the sample spot number for a particular dataset into the Sample entry on the spectrum Display contents window or using the up and down arrows to step through the available samples this automatical
448. s 2 lon Finder will look between and including 999 and 1001 Chapter 19 lon finder Accessing the feature From the base window File menu select Export and from the sub menu select lon Finder Z pi4r_msmso0004 MALDI MS olx File Edit View Instrument Automation Processing Help Open Ctri O Display Spectrum Profiles 1 Masses 12 1040 1 gt SEVE GFS Save As Open Auto Experiment Results Comments Parameters Print Ctrl P Print Repore Print Preview Print Setup ASCI ER Window as a MetaFile Selected Tile as a Metafile m2XML File mzData File Intensity mapping as KSG Ton Finder Biomap Figure 19 1 Export options on the File menu The lon Finder window is displayed jolt Finder x m Ion search template Mass Tolerance Add entry Paste data Import data Remove entries Remove all m Results file destination Eolder Folder file Filename I Auto select filename Dialog data Load new Save Process Figure 19 2 lon Finder window Introduction 319 320 Chapter 19 lon finder Importing a list of masses You can import mass data from a simple ASCII delimited text file or pasted from another application for example Microsoft Excel via the clipboard Text files The text file must be compatible with Microsoft Notepad It must contain at least
449. s account which contain MALDI MS data as a standard Windows tree list as used in the Explorer A next to the folder indicates that the folder contains sub folders i e the list can be expanded further Double clicking the mouse SELECT button on a folder displays all of the data files in that folder in the lower panel of the window If the name of a piece of data is known or part of the name but the user cannot remember the name of the folder in which the data was stored Type into the Find entry any characters to match with stored file names e g the template pr would match PRIMEOOO1 and 09 would match PEG1009 Press the Find button to list all matches The find template is case insensitive and wildcards may not be used When the search is completed all matching datasets are displayed along with the folders in which they were found Selecting a dataset from the search list will cause that dataset to be entered into the currently selected slot on the Load data window Loading data Chapter 8 Loading and unloading data The Data browser window Figure 8 3 gives a brief summary of the datasets stored within each folder To load a dataset from the list simply double click the mouse SELECT button on the desired dataset The Filter Options in the centre of the window may be used to restrict the list to display only runs which match specific operating conditions For example set Flight path to Linear to list only data
450. s are Subtract Simply turns on or off baseline subtraction Width The baseline subtraction algorithm can be viewed as the filtering of the very low frequency information from the spectra This parameter effectively determines this lower frequency Basically the higher the value the lower the cut off frequency The calculated baseline may be viewed by selecting the option to view the baseline from within the Graphs tab of the Display Options window In this case it is displayed on the averaged trace Figure 16 6 It is correctly set when then baseline curve smoothly follows the overall shape of the spectrum without intruding into the peaks themselves The software once again provides an easy way in which to correctly set the baseline Simply place a pair of cursors around the feature in the spectrum normally a single peak which should be retained i e the baseline will not intrude into this feature and import these values into the peak cleanup window by using the button in the base parameters section labelled with the cursors icon Note that setting the baseline width to less than that of the peak will erroneously distort the peak itself Subtracting the baseline 243 Chapter 16 Cleaning up data The baseline being subtracted is shown on the averaged trace 100 80 baseline 60 40 20 0 lnt 5682 1 100 80 5726 6 60 40 20 0 5670 5680 5690 5700 5710 5720 5730 5740 5750 5760 Mass Charge Data after baseline sub
451. s been collected using a fixed aim having already found a sweet spot but with the power scanning it is also possible to choose a power level from the chromatogram by placing a cursor at the point on the graph where power is considered optimum and pressing the cursors button on the power line of the Laser Firing window Displaying Chromatograms Chapter 17 Viewing the collected data Using chromatograms to locate peaks If the chromatogram is simply to be used to locate the sweet spot then data need not be stored as once the sweet spot has been found the chromatogram data can be discarded However other uses of the chromatogram displays require that the profile data be stored Storing data provides the ability to manipulate and re process the chromatograms as a tool for the location of peaks of interest within the data Set Displays to Chromatogram The Display contents window for chromatograms is shown in Figure 17 10 Av Chromatogram Contents x Dataset Trace Sample 1 angio2_msms000Gixg Fa G7 Smoothing Segments Tisai 3 Front Intensity Profile Mass Average Largest Figure 17 10 Chromatogram Display contents window The dataset for which a chromatogram display is required should be selected using the Dataset menu also the Trace and Sample should be selected A chromatogram can be divided into a specified number of segments a single segment will show a single chromatogram over the range o
452. s iTRAQIR Completed E C Documents and Settings bill KRATOS My Documents iTRAQIR Completed E C Documents and Settings bil KRATOS My Documents iTRAQIR Completed E C Documents and Settings bill KRATOS My Documents iTRAQIR Completed E C Documents and Settings bill KRATOS My Documents iTRAQIR Completed C Documents and Settings bill KRATOS My Documents iTRAQIR C Documents and Settings bill KRATOS My Documents iTRAQIR C Documents and Settings bill KRATOS My Documents iTRAQIR C Documents and Settings bill KRATOS My Documents iTRAQIR C Documents and Settings bill KRATOS My Documents iTRAQIR C Documents and Settings bill KRATOS My Documents iTRAQIR AC Dacumante and Sottinacthill VD ATASIMy DacumeantebiTD AMID xl PPPppp P Estimated time remaining 9 seconds aa canci l 3 On completion the Batch Process Complete window is displayed Batch Process Complete x e J Batch process job has successfully completed 4 Click the OK button Using the Batch processor 543 544 Chapter 34 Batch processor XML export The results appear in the required folder for example t Batch processor results olx File Edit View Favorites Tools Help ay Back O bi 2 Search Folders 3 E gt lt ig EK Address D C Documents and Settings ibill KRATOS My Documents Batch processor results l 5 Go Folders Desktop i 3 75 00 07 29 1 109 0001
453. s is a peak detection method used to identify monoisotopic peaks and is used in conjunction with the parameters set within the Peak picking tab The Double threshold feature uses a low threshold to identify all peaks and an upper threshold to identify candidate monoisotopic peaks The Double threshold is based on processed data All data values are examined and the maximum minimum and non zero values are found The formula maximum minimum non zero minimum values is calculated as the number of bins time slots or channels for histogramming data The data is then histogrammed into this number of bins Plotting X bin density verses summed bin count gives a characteristic knee plot rt The lower threshold is the lowest non zero bin intensity and the upper threshold is the intensity of the bin at the knee this is calculated as the point where the diagonal of the graph intersects the plot Double threshold Chapter 16 Cleaning up data The algorithm then looks for supporting peaks that are one Dalton adjacent to the candidate and above the lower threshold The number of supporting peaks is determined by the Maximum isotopes field The lower threshold is not shown on the spectrum Upper 534 28 threshold 60 40 20 soo 805 Sto S18 520 525 530 935 S40 The Mass ranges field allows you to divide the spectrum up in to segments within each segment the Double threshold fea
454. s not normally required Select the I nitialise stage button to move and set the stage to the 0 0 and end stop datum positions No further user action is required at this point simply wait a short time until it can be audibly detected that the stage motors have completed the action 2 Align plate This process allows for possible minor manufacturing variations on a particular plate or for errors caused by inserting plates differently or for fine tolerances on the sample carrier mounting The plate is aligned using the three plate alignment reference points All three reference points must be aligned for correct operation of the stage Values in the Plate X Plate Y Stage X and Stage Y values can be modified by double mouse clicking on the required cell typing in the new value and pressing the Enter or Return key The values in the Stage X and Stage Y columns are derived from the current alignment which is stored in the instrument s EEPROM Sample plates and plt files 29 130 Chapter 13 Preparation for data collection The window has two tool bars which have tool tips Stage Control and Alignment References the functions available are described in the tables below Stage Control Table 13 1 Stage Control functions Icon etrs Sample plates and plt files Action Move the stage The manner of the motion depends on how long the button is held down and whether the Shift key is depressed A single short press
455. s thaliana thale cress 52598 sequences Timestamp 1 Mar 2006 at 10 51 08 GMT Top Score 134 for Mixture 1 gi 9294498 gi 7529717 Probability Based Mowse Score Ions score is 10 Log P where P is the probability that the observed match is a random event Protein scores greater than 60 are significant p lt 0 05 Number of Hits N N N N N N N N N A N 100 150 Probability Based Mowse Score Concise Protein Summary Report Format As Concise Protein Summary Help Significance threshold p lt 0 05 Max number of hits 20 Re Search All Search Unmatched 1 Mixture 1 Total score 134 Expect 2 1e 009 Queries matched 22 Components only one family member shown for each component gi 9294498 Mass 37174 Score 74 Expect 0 0021 Queries matched 11 aldose 1 epimerase like protein Arabidopsis thaliana gil 7529717 Mass 38516 Score 60 Expect 0 054 Queries matched 11 fructose bisphosphate aldolase like protein Arabidopsis thaliana Mascot PMF searches Chapter 18 Protein peptide analysis using Mascot search engine Mascot MS MS searches The main difference between an MS experiment and an MS MS experiment is that the peptide ions from the sample collide with a collision gas as they travel up the flight tube causing fragmentation From these fragments it may be possible to identify the amino acids and their sequence in the peptide sample Typically precursors up to 3 000 Da will produce usable frag
456. s the Calibration window select the Calibration label from the tree as shown in Figure 15 11 below Method Editor default mtd 1 x Sample Method Sample Method default mtd Wy Parent E ME Acquisition Calibrant references v Rast A npn List references Reference editor Compounds 3 Laser Firing Mass Formula Abundance Cursor mass FM Processing 54 C50 H72 N13 012 VIL Calibration 1533 86 C76 H113 N18 016 E Peak Cleanup 1800 94 C87 H126 N21 021 i Monoisotopic Peak Picker E s aa Cas aaee te Goes is MIB Peak Fitering 3657 93 C167 H258 N47 046 Via Mascot J Applications Cursor mass 0 0000 Insert Delete amp Compare Sequence tl inset _ Delete O Oligo Analysis Mass 1534 8500 ae ete ite My Output Formula C76 H113N180 Calculate fAverace gt Vi Print Average Eas O Export Calibration Q Report Calibration 1 lon Finder Tolerance fro ba x tl Fit through zero MI MS MS VQ Acquisition Resolution for Distributions 1000 SE Peak Selection MRH Raster E Dutput Calibration J v 3 Laser Firing ae ZY Processing Select default calibration file EK Peak Cleanup Callin Monoisotopic Peak Picker VIE Peak Filtering VS Mascot My Output X gt Data Storage Get Manual Settings Save Method Load Method Figure 15 11 Method Editor Calibration Calibrant reference files can be created loaded and saved as described in
457. set currently selected for processing If the dataset can be changed on the sub window it will automatically reflect the change on the Spectrum contents window Choosing stacked or overlaid views Spectra from different datasets and or different traces can be displayed in two ways either overlaid superimposed one on top of another so that the baselines are located on the same x axis Displaying Spectra 271 272 Chapter 17 Viewing the collected data or stacked in an isometric projection These two views are selected from the Spectrum Contents Figure 17 4 shows the type of display obtained with each option 1046 5 1046 5 1047 5 1047 5 1048 5 14s Ae 1049 5 1047 4 1048 5 1049 5 o 1046 1047 1048 1049 1050 1046 1047 1048 1049 1050 Stacked traces Overlaid traces Figure 17 4 Example of stacked and overlaid traces Pressing the Apply button updates the selected display Choosing the profiles to display The range of profiles to display is entered in the Profile entry on the base window as either an individual profile number such as 24 or a range such as 24 36 for all profiles between profile twenty four and profile thirty six press the keyboard Return key after entering the value or range of values The whole range can be viewed by simply entering a hyphen without any profile number Alternatively all profiles from twenty four to the last profile can be viewed by entering 24 and all
458. shold may follow the peaks rather than following the noise Subtracting the baseline Chapter 16 Cleaning up data Peak detection The process of peak detection is performed on the processed data after the baseline has been subtracted and any smoothing carried out on the data The software supports two main methods for peak detection as follows e Gradient In this method of peak detection the software looks for the start of a peak indicated by a zero second differential i e d y dx 0 Once this has been found it is assumed to be the start of a peak and the software then looks for the end of the peak by looking for another location where the second differential is zero Next the candidate peak is checked for being greater than the minimum peak width specified in the Peaks parameters and finally checks that the minimum value of the second derivative between the start and end of the peak is less than the value of the parameter rejection peak width Hence this requires that peaks are tall and narrow in shape or at least have an acceptable height to width ratio Threshold In the threshold method of peak detection the start of a peak is determined by the signal after smoothing and baseline subtraction rising above the threshold value indicated The end of the peak is determined as the location where the signal falls below this value once more Figure 16 8 Once the start and end of the peak have been determined the peak is s
459. si0a7 1071 A 1 Select display to be used as an insert 1046 51 1070 1 c Mass Charge sine Insert to go here 2 Position the pi cursors to mark the 80 a boundary of the new insert 40 20 o 100 200 300 400 500 600 700 800 900 1000 1 c F Mass Charge 4 J ee lam 68 34 Mam 7 40 yg angio2_msms0006 MALDI MS of x File Edit View Instrument Automation Processing Help S e 2 5 de E disp spect Z Profiles fi Masses fosron ER 1055 1070 1 c Mass Charge 3 Press Shift and click the i button to create the insert 1000 1 c t Mass Charge gt dm 295 88 mam 1 98 7 Figure 20 25 Creating an inset display 368 Copy insert and delete displays Chapter 20 Managing Data Displays Inserting an inset of the currently selected display into itself can be achieved by the above method or after positioning the range cursors as above by selecting Insert from the Display menu and choosing Inset The Display menu is obtained by clicking the mouse MENU button on the selected display Figure 20 26 Paste Coum Delete gt Row Annotate Display Settings Tags Peak labelling gt Figure 20 26 The Display menu The selected display will appear as an inset Figure 20 25 within the original display The display containing the inset is referred to as its parent display Once an inset has been created the data within the inset can changed by c
460. sis vI Output We Print Dialog data j Wa Export vU Report Loader me I MS MS SY Acquisition BE Peak Selection a Ea Raster 3 Laser Firing I Processing EEX Peak Cleanup lita Monoisotopic Peak Picker ES Peak Filtering 18 Mascot My Output Data Storage et Menaal settir Save Method Load Method Figure 15 30 Method Editor lon finder For each peak of interest lon Finder examines the spectrum and extracts its intensity mass area The results are presented in a text report To use this feature see Ion finder on page 317 200 Method Editor Chapter 15 Automated operation MS MS Peak Selection The Method Editor Peak Selection window provides functionality for identifying how peaks are selected for MS MS processing To access the Peak Selection window select the Peak Selection label from the tree as shown in Figure 15 31 below Method Editor default mtd Sample Method Sample Method default mtd Peak Selection for MS MS Acquisition Vy Parent vI Acquisition M fa Raster X Auto Quality 3 Laser Firing Processing VIL Calibration EZ Peak Cleanup IME Monoisotopic Peak Picker VIE Peak Filtering VIS Mascot JEJ Applications amp Compare Sequence Oligo Analysis V Output YQ Print 3 Export U Report a lon Finder Via MS MS v 3 Laser Firing Vy Processing EK Peak Cleanup v W Monoisotopic Peak Picker VEA Peak Filtering
461. sms0006 MALDI MS BEE File Edit Yiew Instrument Automation Processing Help BIR e 2 Bl lt Defen z Potes v Massesfi1039 ll l S E Display toolbar _ _ __ gt x hall Lil 4 E 75651 931 85 af i3800 p StattiS bar 343 28 1 138 31 me a oa shod oho 2 904 41 h 2 aala 784 95 1002 504 62 Sp 0g 185 19 297 40 p29 4 707 853 37 414 25 iv at egz 7038 Uaa Mih haa A Aaah haddetign eg Neti nitty Val easly 300 400 500 600 700 mz Figure 17 1 MALDI MS window display area Chapter 17 Viewing the collected data Selecting the type of display required The display area is capable of showing both graphical and textual reports including spectra of single or averaged profiles chromatograms intensity variation over profiles calibration curve displays and simulations of peak shapes based upon isotopic distribution It can show the peak positions of calibrant reference peaks and has many advanced viewing features which make it an extremely powerful tool in data manipulation The type of display required is selected using the Display option For each type of Display Spectra Chromatogram etc there is a separate Display contents window with options pertinent to the type of graph or text report chosen The different Display contents windows and the options available on them are covered in each of the following display sections Table 17 1 summarises th
462. sor XML export Adding files to the Batch Processor There are several methods available to you Drag and drop You can drag and drop a directory or selected files in to the Batch Processor window only the run files move 1 Open Windows Explorer and navigate to the required directory 2 Drag and drop the directory or selected files in to the Batch Processor window Adding a selected run file 1 Open Windows Explorer and navigate to the required run file 2 Right mouse click on the run file Open Print Add to Batch Process job Convert to Adobe PDF Convert to Adobe PDF and EMail Scan for Viruses Open With gt Send To Cut Copy Create Shortcut Delete Rename Properties 3 Select the Add to Batch Process job menu item the run file is added to the Batch Processor 4 Repeat for any other required run files Using the Add button 1 Select the Add button the Open window is displayed 2 Navigate to the required run files usually within the Data folder c Program Files Shimadzu Biotech Launchpad Data Using the Batch processor 541 Chapter 34 Batch processor XML export 3 Highlight the required files you can use the Ctrl or Shift keys in conjunction with the mouse to select deselect files Open i Lx Look in E Rag 2 1 117 114 x l ea gt HS Fe 97 00 09 45 3 5 00 07 97 00 09 48 My Recent 98 00 09 51 Document
463. ss Charge 100 110 Mass Charge Figure 20 38 Adding an arrow line ona graph 386 Annotation Chapter 20 Managing Data Displays Adding text annotation Text is placed in a display by pressing the E button The cursor in the display will change to a flashing tri e cursor indicating that the cursor is in text mode Click the mouse SELECT button on the display at the position the text is to be inserted Type in the text for the annotation and press the keyboard Return key Figure 20 39 Use the same method for boxed text The annotations will move with the scaling and panning of the graphs because of this the annotation text lines and boxes must lie within the range 0 100 intensity Annotations will not be permitted outside this range Click Ba and type in the required text Angiotensin II fragmentation 100 Mass Charge Annotation 387 Chapter 20 Managing Data Displays Figure 20 39 Adding text to a graph Annotation with a boxed region A region of interest on a graph can be highlighted by the use of a box A box is created in the same way as annotation lines however in this case the two cursor cross hairs delimit the top and the bottom of opposite corners of the box Figure 20 40 Position the cursors so that the two cursor cross hairs mark the diagonal corners between which the box will be drawn On the New Annotation window select A box will be drawn between the cursor
464. ssing the OK button and confuted by depressing the Cancel button Sample plates and plt files 31 Chapter 13 Preparation for data collection Raster laser firing Defining a sample raster for acquisition Acquisition default_linear Firing Exp Tech Auto Quality Storage Slide Raster Tuning Save Raster Type Regular Rectangular 7 Raster Style TY Raster pa um Height 116 00 um Centrex 0 0 um Centre 00 um Spacing 10 000 um Points 129 Calculate Raster Based On Spacing IV Width 96 00 Figure 13 12 Defining a ane raster to control laser iring You can define a raster which can be applied about a sample well to govern the laser shot pattern Two types of rasters can be defined regular rasters and free hand rasters the choice is selected from the Raster drop list box The regular raster is set up by defining the regularly spaced distribution in microns of a number of shots about the centre of the raster the centre point X and Y define the centre of the raster relative to the centre of the well to which it is applied Width and height define the extent of the raster Raster laser firing Chapter 13 Preparation for data collection If the option based on spacing is selected then the number of shots will be calculated when the Calculate raster button is selected If the number of shots is entered and based on spacing is not selected then the spacing will be calculated TV ras
465. st peaks in the mass range This is achieved using the display linking feature Figure 20 49 shows two data displays a spectrum and a mass list With the spectrum display selected move the mouse pointer over the mass list press and hold down the keyboard Alt key and click the mouse SELECT button The mass list instantly changes to show a listing of the peaks in the same mass range of the spectrum A green border is drawn around the linked display This border is only shown on screen it will not appear when a print is made Parameters for the mass list for this example are set so that only the 10 largest peaks in the mass range are reported Linking data displays Chapter 20 Managing Data Displays J pep_mix_2466d0005 MALDI MS oix File Edit Yiew Instrument Automation Processing Help g JE 2j E x Displey Specttum v Profiles 1 Masses 1525 1546 l gt I Mass Area Total Apex m Resolution SIN Int 6 7 m sum 955 mY Profiles 1 142 Smoq 1525 64 0 35 O47 0 01 624 54 0 23 1528 27 030 0 45 0 02 2836 40 1 53 1529 40 014 0 07 0 01 3007 64 0 52 1534 33 1530 27 0 19 0 09 0 02 3025 05 1 03 J 100 1435 30 1534 33 100 00 49 27 673 2901 71 811 90 y 1535 30 9214 45 39 6 30 2911 61 761 44 90 1536 34 962 4 74 116 285929 138 90 1540 44 015 0 07 001 2714 22 0 80 154461 010 0 05 0 01 2746 28 0 00 80 70 60 50 40 elected display black border Linked display green border 30 20 6 34
466. storing data laboratory notes may be added to the data Laboratory notes may consist of any textual information whatsoever These notes could relate to sample and or matrix preparation or other information They will be kept with the data at all times Up to ten note files can be created of virtually unlimited file length Notes may not be added during data collection To add notes to data or display notes which have previously been created set the Display type to Notes and then press the toolbar display contents button to show the Display contents window for notes Figure 17 17 ba Notes Contents BEI Dataset 1 angio2_msmsO00thd Note ia 2 3 4 5 6 7 8 3 10 Other Browse Edit Delete File angio2_msms0006 notel Figure 17 17 Display contents window for notes Creating Notes for data The dataset to which the note files are to be attached should be selected using the Dataset menu Notes 1 to 10 specify up to ten note files belonging to that dataset To create a new note file e g note 1 for the select dataset select Note 1 then click on the Edit button The Windows Notepad editor will be displayed allowing the notes file to be edited The notes are of arbitrary length and are stored as standard ASCII text files Displaying laboratory notes Chapter 17 Viewing the collected data When you have finished creating the note file select Save from the File menu in Notepad and press Apply
467. strument Calibration Cytochrome C To create a calibrant reference file press the Reference editor button on the Calibration window The Reference editor window will appear Figure 27 3 af Reference Editor 1 x Reference file selection Filename TOF2_mix List Save References Formula Abundance Angiotensin 2 Angiotensin 1 Gu 1 fib N acetyl renin ACTH_1 17 ACTH_18 39 ACTH_ 38 Edit reference Mass H 046 5422 Formula ngiotensn2 Compounds Update Mass Resolution Calculate Moncisotopic X l 6000 Figure 27 3 Reference Editor window 462 Introduction Chapter 27 Instrument Calibration Creating a new reference file Type in a reference file name File name for the new file up to 20 characters Select the instrument polarity Mode for which the reference file will be used This can be any of the following Table 27 1 Instrument Polarity Options Any Any Lin Lo Lin Lo Lin Hi Lin Hi Ref Lo Ref Lo Ref Hi Ref Hi where indicates Positive Negative ion mode Lo Hi Low High mass mode Lin Ref Linear Reflectron mode A general file suitable for all modes can be created using Any Any If the atomic composition of the reference mass is known an accurate molecular elemental weight can be obtained using the formula to mass calculator on the window Type the formula in to the Formula entry Formulae are entered with
468. sts in the Windows System folder Error 19010 Unable to get the dzip function address from the Dzip32 dll Unable to open the specified dll file Check that Dzip32 dll exists in the Programs directory If not copy the file from the software installation CD Error 19020 Unable to get the dunzip function address from the Dunzip32 dll Unable to open the specified dll file Check that Dzip32 dll exists in the Programs directory If not copy the file from the software installation CD Error 19030 Unable to load the Dunzip32 dll Check that this file exists in the Windows System folder Check that Dzip32 dll exists in the Programs directory If not copy the file from the software installation CD Error 23000 There is a problem with one of the analyser turbo pumps Please call service Press OK to reset the instrument Error 23010 There is a problem with the SAC turbo pump Please call service Press OK to reset the instrument Error messages Chapter 44 Summary of error messages Error 23020 There is a problem with the SAC gauge Please call service Press OK to enter standby Error 23030 The HT interlock has operated Please call service Press OK to continue in standby Error 23040 There is a problem with the analyser gauge Please call service Press OK to continue pumping Error 23050 Could not open gate valve Please call service Press OK to continue Error 23060 Could not close gate valve This error is only applicable when the Axi
469. t lt Type enumeration gt regular freehand lt NumPoints unsigned integer gt lt Path enumeration gt tv serpentine lt Point float microns float microns gt For regular rasters the Point values are not required as they are calculated based on width height and number of points Multiple Point values are allowed up to 100 Block lt MSx gt lt Acquisition gt lt AutoQuality gt lt MonitorRange float lower mass float upper mass gt lt NoiseStart float mass gt lt NoiseWidth float mass gt lt PowerLimits unsigned lower power 0 180 unsigned upper power 0 180 gt lt StartPower unsigned power 0 180 gt lt Prescan enumeration gt true false lt PrescanProfilesPerPt unsigned integer gt lt MinPoints unsigned integer gt lt CutoffPercent unsigned integer 0 100 gt lt MinI ntensity unsigned integer mv 0 2000 gt lt MinResolution unsigned integer gt lt MinSN unsigned integer gt lt MinSNPercent unsigned integer 0 100 gt lt MaxRejects unsigned integer gt lt LockMassEnable enumeration gt true false lt LockMass float mass gt lt LockMassTol float gt lt LockMassUnits enumeration gt Da mDa ppt ppm ASCII Text Method file format Chapter 15 Automated operation Block lt MSx gt lt Acquisition gt lt LaserFiring gt lt Configuration string gt configuration or mode name must be accessible in parameters lt MassRange floa
470. t corner Data File m Grey square You display spectrum and Mascot data within another tile When you select the required data the Tile Manger is displayed to allow you place the new typically alongside the experiment results ws 417 4E0023_0001 H18 5 Refer to the Getting started guide Chapter 2 Using the Axima Launchpad amp MALDI MS section Multiple tiles To display the Tile Manager 1 Move the mouse pointer over the square it changes to this Pi 2 Double click the mouse left button Current Tile Layout i Cancel Insert Row Column Inset Display Settings Delete Row Insert a row or column Column Inset All Insets 3 Select the Insert gt Row or Insert gt Column buttons see the following images for examples Using Auto Experiment Results viewer 229 Chapter 15 Automated operation Spectrum display The following image shows a spectrum of the selected data displayed in a left hand column 4 0023_0001 318 1881_0001 AE0023_0001 H18 969_0001 MALDI MS File Edit View Instrument Automation Processing Help bel Jae A 8 x a4 Display Spectrum x Profiles 1 16 Masses 1459 2426 14 gt lnt 20 mY 0 0 mV 1881 87 100 Data Acquisition 80 Data Acquisition CID on 968 52 Data File 60 a C Program Files Shimac 40 16 08 2006 14 28 50 Data Acquisition 1866 27 CID on 1251 74 Data Acquisition CI
471. t low mass float high mass gt lt Calibration string gt acquisition calibration name must be accessible lt Power unsigned integer 0 180 gt lt Profiles unsigned integer gt lt Accumulation enumeration gt 1 2 5 10 20 50 100 200 The following parameters only have meaning for the Axima Confidence Assurance instruments and will be ignored if specified for the Axima QIT instruments lt Neutrals enumaration gt true false lt Gate enumeration gt off on blank lt GateLow float lower mass gt only lower mass used for blanking lt GateHigh float upper mass gt lt PulsedExtract enumeration gt off on lt PulsedExtractMass float mass gt The following parameters only have meaning for an Axima QIT instrument and will be ignored if specified for the Axima Confidense Assurance instruments lt AcquisitionMode enumeration gt xlow low mid high xhigh custom lt CustomName string gt only used if AcquisitionMode Custom Block lt MS2 5 gt lt Acquisition gt lt MSN gt lt PrecursorList float precursor1 float precursor2 etc gt precursors 1 through 4 are allowed Only those precursors consistent with the MSn will be used lt GateList enumeration enumeration etc gt 70 250 500 1000 list of values one for each precursor ion specified lt CI DList unsigned unsigned etc gt 0 1000 list of values one for each precursor ion specified lt CIDGas enumeration gt
472. t menu shown in Figure 14 6 below Focus well Expand plate overview Figure 14 6 Pull right menu options available on the plate views Goto location produces the popup menu shown in Figure 14 7 below this enables a particular well centre or any point on the sample plate to be centred in the detailed view Goto well centre or plate location x Locate at centre of well Well code a1 N Locate at plate coordinates Current X location Y location 72 2500 28 7486 Requested X location Y location 72 2500 28 7486 Figure 14 7 Goto location popup menu Focus well is similar to the Goto location option If a well is under the cursor when the pull right menu was activated then the centre of that well will be given focus centred in the detail view Expand plate overview is the third option on the plate pull right menu when selected this produces a larger view of the plate overview which makes well selection easier The expanded view has the same functionality as the normal view with the additional Sample selection 43 Chapter 14 Collecting data from a sample feature that if the mouse pointer is over a well on the plate then the sample id of that well is shown at the bottom of the window If the OK button is selected then the expanded view disappears but the expanded window details are inherited by the normal views of the plate If Cancel is selected then the popup disappears and the normal v
473. t the Match Peaks tab on the Peptide Settings window as shown in Figure 42 32 below amp Peptide Settings Keyboard Display Fragmentation Match Peaks Nested PSD Spectra Search EEE a Rea MEE Y Select peaks Single sequence peak match Report missed theoretical peaks V Report missed experiment peaks IV General Tolerance Da z OK Cancel Figure 42 32 Match Peaks tab of the Peptide Settings window Chapter 42 Sequence Calculator The search can be carried out in the Currently loaded sequence in which case the sequence in the selected panel of the Sequence Calculator window will be searched Specifying Load window selections causes each of the selected sequences in the Load Sequence window to be loaded then digested fragmented and the resultant fragments matched against the selected dataset Where the currently loaded sequence or a single selection in the Load Sequence window has been made the Report missed experiment peaks option can be ticked this causes the report to contain the missed peaks as well as the matched peaks Similarly by ticking the Report missed theoretical peaks option the report will contain the missed peaks that where expected together with the matched peaks Select a Tolerance to be used in the peak matching peaks which are outside this tolerance window will not be matched Press Match and the following operations will be performed The loaded or selected se
474. taches protecting groups to the current sequence in the Compare Sequence panel see Sequence Calculator on page 601 Sets fragmentation options when generating a Sequence report see Sequence reports on page 620 Chapter 15 Automated operation A pull right menu can be accessed via the mouse MENU button click on right mouse button when the cursor is over the Compare Sequence panel as shown in the Figure 15 19 below m 8 Cut Ctrl x YGGFLRRIR coo ae A Paste Ctrl Delete Sequence Find 144 p PP Name Untitled Figure 15 19 Pull Right pt of Compare Sequence pane Table 15 2 Pull Right menu functions Remove the currently selected sequence and Cut place it on the clipboard Copy Copy the current selection to the clipboard Paste Paste the contents of the clipboard at the current insertion point Delete Deletes the current sequence in the panel Sequence Find a given sequence within the Compare Find Sequence panel see Finding a specific sequence on page 614 A search can be carried out in the Currently loaded sequence in which case the sequence in the panel of the Compare Sequence window will be searched Specifying Load window selections causes each of the selected sequences in the Load Sequence window to be loaded then digested fragmented and the resultant fragments matched against the acquired dataset To define the peak window in which to compare the sequence select the Se
475. tation window example in Figure 42 28 Annotation ot x Display labels list Type Y Dataset All datasets x Trace fal Traces X Sort Increasing mass 7 F 2 175 21 default 0 Processed F 3 288 37 default 0 Processed F71 712 83 default 0 Processed Load Include Save Properties o ces Introduction 631 632 Chapter 42 Sequence Calculator Figure 42 28 Peak markers generated from peptide PSD fragmentation When enzymatic digest has been selected markers generated by the Peptide calculator are marked with the fragment number to which they belong e g markers from fragment 1 have the suffix 1 from fragment 2 the suffix 2 The digest fragments themselves are marked as F fragment e g F 1 and F 2 These assist in the identification of the enzyme digest product molecular ions in the spectrum Calculating spectra of theoretical fragmentation results Introduction We have already discussed under Displaying simulated data in section on page 415 application of a Gaussian fit method to the isotopic distributions calculated for a molecular formula and the formulae of up to four adducts to yield a theoretical spectrum at given resolution conditions Here that method is extended to computing the spectrum for the fragments produced by enzyme cleavage and or PSD fragmentation of a peptide as described earlier in Sequence reports on page 620 It should be noted that as we are usu
476. te Profiles to File rOWSE Acquiring sample G14 Acquiring profile 1 of 100 Figure 14 4 Laser Firing on the Axima Performance Acquisition positive Be xi Firing Exp Tech Auto Quality Storage Slide Raster Tuning QrrT ToF ms T Auto quality Profiles a per sample aaa Shots fe x accumulated per profile MSn Precursor Ions Mass Range E a HA 1005 300 509 2k 3k Sax 8 aa ix I Activate Accumulation Filename Accumulate Profiles Figure 14 5 Laser Firing on the Axima Resonance The figures above show two views of the sample plate The larger area shows an overview of the plate with all 384 wells represented The smaller area shows a more detailed view of part of the plate The location of the detail view on the overview is Sample selection Chapter 14 Collecting data from a sample indicated by a rectangular box The well location codes a combination of letters and numbers by default are shown on the detail view only The detail view can be zoomed in or zoomed out with the buttons Well Al is towards the upper right hand corner of the plate and H12 towards the bottom left In Figure 14 4 only two wells A1 and B1 are selected indicated by the well being block filled Individual wells are selected and de selected using the left mouse button All wells can be selected or all de selected using the buttons Clicking the right mouse button over any well produces the pull righ
477. tech Launchpad Parameters c Highlight the required file d Select the Open button the Open window closes and the selected file name and path appears with in the Legacy File Settings window 4 Select the OK button Using the Batch processor Chapter 34 Batch processor XML export Using the command line editor Introduction You can use the Windows command line editor to run a command line application run2xml to convert MALDI MS run files into mMxXML or mzDATA files cx Command Prompt Of x Microsoft Windows XP Version 5 1 26661 lt C Copyright 1985 2661 Microsoft Corp C N Figure 34 8 Command line editor The run2xml application is intended for use by users who are familiar with e basic command line commands to navigate through a directory structure using wildcard characters We recommend that you run the application from the directory in which the data files reside However you can you run the application from any directory and use the i or o switches to set the input or output directories Using the command line editor 547 548 Chapter 34 Batch processor XML export Example usage Typical format of a command is run2xml lt filespec1 gt lt filespec2 gt lt filespec3 gt mzxml mzdata OPTI ONS Single file conversion example To convert the file 54 00 05 28 1167 0001 run to mzxml data format type in the command 1 In the command line editor
478. tech Launchpad x a Fe Calibration References Comments stage My Recent Data E License txt Documents Databases E Experiments Export Desktop Filters A Internet a Labels r vi Log My Documents Methods Parameters y ig Peptides My Computer pormers Programs i Ets File name z Open Places Files of type Text files txt ba Cancel Figure 17 19 File selection window for Notes files After selecting the text file to use press Apply on the Notes Contents window 292 Displaying laboratory notes Chapter 18 Protein peptide analysis using Mascot search engine Chapter 18 Protein peptide analysis using Mascot search engine 293 294 Chapter 18 Protein peptide analysis using Mascot search engine Introduction Introduction You can search a database of proteins using the Mascot search engine to aid your analysis of a spectrum The Mascot database search engine typically resides on the Matrix Science web site www matrixscience com and you can access it over the Internet However your organisation may have a Mascot facility installed on an internal server which you can access over your local area network This section assumes that you have access to either system This section describes two types of analysis MS or PMF Peptide Mass Fingerprinting to analyse a spectrum of a digested protein Peaks relate to the masses of the peptides formed from the digest The result
479. ted 1 out of 1 matches Save Sequence x Save in C Peptides Her B Import Sequence Look in gt Peptides fe Desktop E ACTH ksq E angiotensin 1 ksq 5 angiotensin 2 ksq E E angiotensinogen ksq Desktop 2 default ksq rey Fa ptar ksq My Documents My Documents sr ar e e My Computer Figure 5 17 Sequence Calculator windows File Processing menu 5I 52 Chapter 5 Window and menu guides Sequence Calculator File Edit view Settings Lol Hej gt gt Generate Peak Markers Length 0 Synchroni p ai Calculate Name Untitled N terminus Hydrogen H Moncisotopic v terminus Hydroxy H O J Fragmentation Singly charged Y ation Proton H x Digest amp Peptide Settings Keyboard Display Fragmentation Match Peaks Nested PSD Spectra Ala Arg Asn Asp Cys CysOx _ Gin Glu Gly His te _Leu tys Met Om Phe Phsr Pro Ser Thr Tp Tyr Val Peptide Settings Digest fragments Method Trypsin Report Fragment number V Position M Masses v Bull Breese indices M HPLC indices Vv Elemental Formulae IV Sequence Vv Sort by Mass ha Missed cleavages fo Mass limits 0 2000 Keyboard Display Fragmentation Match Peaks Nested PSD Specta lonisation fragments
480. ted while the Archiver window is open then the user should force the Archiver to refresh the window contents by pressing the Refresh button Any file selections made will be lost so this should ideally be used prior to selecting files for archive Archiving to a file To archive data to a file instead of to a floppy disk follow the same procedure as above but instead of inserting a floppy disk or other removable media select a fixed drive location and folder from the Archive to file window Figure 32 5 This could be on the local disk or over a network anywhere registered as a valid drive location in Windows This file can then be backed up or archived to tape or other media using the normal backup procedure Restoring archived data Introduction To restore archived data from removable disk media place the last disk of the archive set in the drive and press Restore from By default the archiver will look for a floppy disk in drive A if one is not present a message warning that drive A is not accessible will be displayed Either insert a disk in A and press Retry or to use another removable media drive letter select Cancel and select the new drive from the Save in list Restore archive from file 21x Look in E My Documents vl e ex EJ Axima TOF2 service guide a Axima TOF2 service guide zip FM template FE test zip My PSP8 Files Files of type Zip file zip ki Cancel Figure 32 7 Restore archive from file windo
481. temporary list of reference masses can be created for the purpose of calibrating the instrument Type in the mass of the reference compound into the Mass entry and press Insert or use the Compounds button which allows compounds defined in the Compounds Database to be loaded Figure 27 4 Collect a number of profiles from the calibrant sample spot to obtain a characteristic spectrum of the reference compound When the instrument is attempting a calibration it will compare the position of the peaks obtained with those in the reference list E Calibration Ble ka Calibrant references gt gt Calibration Files Dataset None Reference editor Compounds Name tof Save Mass Formula Abundance Cursor mass Time Load named calibration 7 Load r Fragment fit Parent mass fo oooo ee setup Apply Remove Calibration I Auto calibrate Fit through zero M Correct Load Save Cursor mass ee Insert Delete Tolerance 400 J moa 7 t Mass j istribution Resolution 10000 KESOIUUOIT Formula Calculate average x 10000 Calibrate Undo Combined Cal Figure 27 6 Controls on the calibration window Calibration of the instrument 467 468 Chapter 27 Instrument Calibration On the Calibration window the Dataset which is to be calibrated is displayed in the top left hand corner along with a colour box indicating the colour of the process
482. ten than not in the routine analysis of polymeric biochemical and pharmaceutical compounds specific reagents species or amino acid regularly crop up time and time again It would be useful to be able to create shorthand forms of notation which could be used throughout the software suite to represent these regularly occurring groups The Compounds Database provides just such a tool It allows a database containing user defined groups and species to be created To start the Compounds Database editor select Compounds Database from the MALDI MS programs menu on the Taskbar Figure 38 1 Launchpad BBE Show on startup V _Heb Click the Compounds database icon T JB cl aol MALDI MS Search Archiver Enzymes SaL Or use the Start menu system enana Eeka resi Programs Documentation we a fa Archiver Compounds Database Accessories lt Documents a i 2 Configure Environment Ag Administrative Tools Element T i P gt G Shimadzu Biotech Launchpad kd Settings A Element Database Editor 2 N faa Quadralay a Enzymes FA p Search IT RoboHelp Office ov ae Launchpad rtDraw Help and Support a ma Log Window 7 SoundMAX F3 mars oe a fa 7 Run I Startup pl Polymers g Symantec Client Security Sample Plate Editor 3 Log Off bill Volo View Express gt seach er y z Turn Off Computer Figure 38 1 Starting the Compoun
483. ter 1 Introduction Chapter Introduction Chapter 1 Introduction About this User guide This User guide is available as an electronic publication in PDF format only and is supplied on the CD containing the 2 9 software Part number 97 344R11 Description Launchpad 2 9 software User guide It is a comprehensive guide that details all the features and how to use them Change history This guide has the following change history Issue Date Change Issue 12 Oct 2009 Revised for 2 9 1 software Issue 11 March 2009 Revised for 2 9 software Issue 10 Oct 2007 Revised for 2 8 software Issue 9 April 2006 Developed for Axima TOF Contact details Kratos Analytical Limited Tel 44 0 161 888 4400 Wharfside f Fax 44 0 161 888 4401 Trafford Wharf Road aX l i Manchester Web sites M17 1GP www kratos com United Kingdom www shimadzu biotech net Email support kratos co uk For details of oversea offices visit the web site s or contact the UK office About this User guide Chapter 1 Introduction Recycling policy All Aximas are marked with the adjacent symbol This means that at the end of its useful life it must not be disposed of with general household waste Contact your local Kratos or Shimadzu office or distributor when the instrument has reached the end of its life and they will advise you regarding its disposal Axima and MALDI MS software The Axima is a MALDI TOF mass spectromet
484. ter Serpentine raster Figure 13 13 Order of laser shots for the two regular raster styles The direction of the raster is set at the Raster style drop list box as either TV raster or Serpentine raster see Figure 13 13 above for an explanation of the order of shots for the two raster styles For a freehand raster only the length and width for a rectangular well or the diameter for a circular well raster are required the actual points are then specified by mouse selection Select the Clear all points button to remove all existing points and begin a new selection Load and Save buttons are provided to access standard dialogues for loading and saving raster rst files The use of raster files to control an acquisition is described in the next section Creating raster files using the ascii2raster utility A utility program ascii2raster can also be used to create a rst file The simplest way to use this utility is in creating a regular raster In this case all of the required information is passed as a set of command line arguments to the utility i e ascii2raster wWidth hHeight xOffsetX yOffsetyY pPoints tType inputname txt filename rst where Width is the raster width in microns Height is the raster height in microns OffsetX is centre point X offset dimension in microns Offsety is the centre point Y offset dimension in microns Raster laser firing 33 134 Chapter 13 Preparation for data collection
485. th any substance which is radio active or biologically active and dangerous unless e Biochemical proteins peptides yes no you e Radioactive yes no e Micro biological living cells yes no e Polymers yes no e Decontaminate the equipment and e Provide proof of decontamination e Dangerous to human health yes no If you have answered no to all of these questions YOU MUST CONTACT YOUR SERVICE go to section 4 otherwise go to section 3 and section 3a CENTRE BEFORE YOU RETURN EQUIPMENT on the reverse side of this form SECTION 3 LIST OF SUBSTANCES USED IN THE EQUIPMENT Are the hazards restricted to the vacuum envelope If no please give precise details of other parts of the equipment that may be contaminated Please add any further information on a separate sheet and send with this form now complete section 3a over SECTION 4 RETURN INFORMATION Please state reason for return Are you making a warranty claim against this return SECTION 5 DECLARATION Name Print Job Title Organisation Phone number Address I declare that the above details are accurate and that I have not withheld any relevant information I have followed the requirements of the returns procedure DOC 102 Signed Date DOC 075 Page 1 2 Issue 7 October 2005 Return Authorisation Number SECTION 3a EQUIPMENT STATUS DECLARATION i Micro biological Substanc
486. the dataset being examined These are Focus well moves the well directly underneath the mouse pointer to the centre of the detailed plate view Expand Plate Overview pops up a larger version of the plate overview useful for ease of selection of wells Moving the mouse over the expanded view causes the sample ID of the well under the pointer to be displayed Any alterations made to the expanded view are inherited by the normal plate view when the OK button is selected Show Test status Show Acquired only appears if the dataset was processed using the Oligo Analysis software see Automated Quality Analysis of Oligomers on page 187 for more detailed information which has a pass fail or uncertain test result The display toggles between showing which wells have been fired at where a green outer border indicates a well from which data has been acquired and showing test result status where green indicates a passed red a failed and grey an uncertain result Focus well Focus well Focus well Expand Plate Overview Show Test status Show Acquired Expand Plate Overview Expand Plate Overview Figure 12 7 Right mouse menu options on multisample plate views Multiple sample datasets 116 Chapter 12 Introduction to displaying data Multiple sample datasets Chapter 13 Preparation for data collection Chapter I3 Preparation for data collection S1I7 118 Chapter 13 Preparation for data collection Introduction
487. the Isotopes option Press Tabulate to generate the polymer series with the selected average molecular mass and desired mass window The Polymers window will display a scrolling table of mass intensity and formulae for each of the peaks found This table can be converted into a reference file which can be loaded into the MALDI MS program to obtain a display of the polymer series Creating a polymer reference file Introduction Having created a list of polymer masses type in a name for the reference file and select the instrument polarity with which it will be used either a positive or negative ion mode reference file Press the Generate button on the Polymers window and the reference file will be created Chapter 40 Polymer simulation Displaying a polymer reference file Having generated a polymer reference file it can be loaded and displayed in the selected display within the MALDI MS base window Set Display to Reference then press the a button to display the Display contents window for reference files Figure 40 4 d Reference Contents BEI Data from EGEAS Reference nylon6 List Traces Profile Peaks Auto mass range V Resolution 2000 Figure 40 4 Settings for displaying reference files On the Reference Contents window set the Data from option to Other reference Select the reference file by pressing the List button and selecting the reference file from the list which appears Select t
488. there is no name LA for the current experiment then the user is asked to provide one as per Auto Experiment e M Chapter 15 Automated operation Table 15 7 Auto experiment experiment icons Import the comments for the current group from an Ascii comments file The required format for such a file is described in ASCII comment files on page 89 above Import an Ascii text file The required format of such a file is described in ASCII text experiment file formats on page 210 below Import a Method format file from an Ascii text file The format of the file is as defined in ASCII Text Method file format on page 214 below The complete experiment parameters are imported including the plate wells definition and the relevant method s applied to these wells Start running the current experiment and optionally generate an experiment results file If sample processing is being carried out pause it If sample processing is paused restart it If sample processing is being carried out or if sample processing has paused abort it Sample Groups Controls A toolbar and list control constitute the sample group controls The toolbar is used to add update and remove sample groups and modify the running order of sample groups Groups are normally run in the order that they appear in the list control and a well can only be processed once in a group For example if a well is selected more than once in multiple groups o
489. thod EL Ga Sample Method defautmd Parent Laser Firing ME Parent BME Acquisition r Setup Raster X Auto Qualiy cocco I 3 Laser Firing B ME Processing Mass Range 1 0 10000 0 Method Editor LE Calibration Auto Experiment Peak Cleant Calibration hd VIE Monoisotopi Stitt WILE Peak Fiterin iTRAQ MR Mascot 5 r B ME Applications Laser Firing ChIP Imaging ExperimenN AA Compare Se v f Oligo Analys ma p 22 be eee a ba A a eei A Print Optional AP Expr Brofies 10 persample n MU Report features and lon Finder Shots fioo z accumulated per profile Vy MS MS instrument E MA Aegiion Laser RepRate 500 Cc Peak Select specific items Vif Raster Ionete off fon Blank 500 0 7000 i p i d VX Auto Quality a ot Bar ty re inr Lait Laser Firin D are e Auto Experiment BE Plate Experiment EB gy Ral 64 OSB B28 70g elas Experiment file name not set Experiment Selection Selection Detail r Comments and Results Title Prefix F Results File p E mail Notification T Upon completion of this experimen send an e mail to the address specified below feet uo x t lt Type Stat End Samples Method Datare Comments 46 Automation menu Figure 5 11 Automation menus a Chapter 5 Window and menu guides LC MALDI BBE Eile Experiment Help SW Prin Precursor Selection Sampl
490. throughput protein ide All parameters are available Scenario 239 240 Chapter 16 Cleaning up data Smoothing collected data Smoothing the data reduces the spikiness caused by transient signals Figure 16 3 100 Original data Smoothed data 100 50 0 a eT a a A a aa 5600 5800 6000 6200 6400 Figure 16 3 Smoothed data using an Average filter Three methods are available for smoothing the data Average Gaussian and Savitsky Golay The average smoothing filter simply moves along the collected data channels adding together a number of channels as specified by width and dividing by that number to give an average signal Figure 16 4 Smoothing collected data Chapter 16 Cleaning up data 7 Smoothed data 2 Original data x Figure 16 4 Example of a3 channel average smoothing ilter The length of the smoothing filter varies linearly with mass starting with a very small filter length at low mass and increasing up to Smooth width channels at the highest mass The Gaussian and Savitsky Golay filters perform somewhat more complex processing of the signal which takes a little longer than the Average filter The smoothing parameters explained briefly are e Average Takes the mean value of the width time channels centred around the current data point and replaces the current data point with this value Gaussian convolutes the data centred around the current data point with a gaussi
491. thyl lect a protecting grou Benzyl selec p g g p Benzyloxy from the list Press the lucreny lncth Add button and the group Trinethylacetenidasethyl t_Butyl will be attached t_Butoxy i t Butvlthio Add to All Remove Remove All For m E E ME ne GLYAPSALVLTMGHGNS 5 10 15 Figure 42 14 Attaching a protecting group Introduction 615 616 Chapter 42 Sequence Calculator Protecting groups are defined in the Compound Database see Defining protecting groups on page 576 To remove a protecting group highlight the unit to which the protecting group is attached select Protecting groups from the popup Edit menu and click on Remove Links Units within sequences can be cross linked to other units by the following procedure First highlight a single unit within the chain as shown in Figure 42 15 then select Links from the popup Edit menu amp Link Sequences m E ME mm GLYAPSBLVLTMGHGNS Link A at position 7 _ Link at position 0 f Loss H Add Remove 5 10 15 1 Highlight a single unit and select Links from the popup Edit menu m E GLYAPSALVLTWIGHGNS 5 10 is amp Link Sequences x Link A at position 7 Loss H Link M at position 12 Loss H Add Remove 2 Highlight a second unit and click on the get selection button 3 Click the Add button a link is created a E E ME GLYAPSALVLTMGHGNS be E 5 10 is Figure 4
492. ting data and data displays 3 Select the required data type radio button and click OK Save As 2 LX Save in Data x e ef Ee My Recent Documents E Desktop 9S My Documents BE My Computer re amp My Network File name angio2 msms0002 X Places Save as type mmexXML mzXML x Cancel 4 The default file name corresponds to the name of the original data You can change or amend it 5 Click the Save button mzData file The mzData is a data format that captures the peak list information The supported version is 1 05 For more information visit the Human Proteome Organisation HUPO Proteomic Standards Initive Mass Spectrometry Standards Working Group web site at http www proteomecenter org Exporting to an mzData file 1 Display the sample profiles and mass range of the data which you wish to export 532 Exporting mz data formats Chapter 33 Exporting data and data displays 2 Select File gt Export gt mzData File Save As 2x Save in E Data amp EE Fe My Recent Documents A Desktop My Documents E hes My Computer Y e URETS File name Places Save as type meData mzData z 3 The default file name corresponds to the name of the original data You can change or amend it 4 Click the Save button X angio2 msms0002 if Exporting mz data formats 533 Chapter 33 Exporting data and data
493. tinue At the Database field select the required search engine from the drop down list MSDB Comprehensive non identical protein database NCBInr Comprehensive non identical protein database EST human Human subset of GenBank EMBL DDB sequences from NCBI EST Division EST mouse Human subset of GenBank EMBL DDB sequences from NCBI EST Division EST_others SwissProt a high quality curated protein database Random Random sequences for verifying scoring statistics Mascot PMF searches 297 298 Chapter 18 Protein peptide analysis using Mascot search engine Reporting Use these options to configure the sections of the report which the server creates for you You may also personalise your report with your name and e mail address Your name your name kratos co uk Arabidopsis thaliana MS experiment SMATRIX SGiuxces Mascot Search Results User Email Search title Arabidopsis thaliana MS expriment The first three fields allow you to personalise the subsequent report as shown in the example above 1 In the Name Email address and Report title fields enter the information you wish to appear at the top of the Mascot Search Results page 2 At the Top hits to report field select the required number from the drop down list Mascot PMF searches Chapter 18 Protein peptide analysis using Mascot search engine Protein identification search criteria Protein identification search
494. tion 26 Chapter 3 Getting started Introduction Table 3 1 Description of the applications Program ek e e fe Function Archiver allows you store files and folders on to mass storage media for example a CD Enzyme editor allows you define a library of enzymes and their cleavage sites for peptide digests Elements allows you to view a detailed Periodic Table Compounds allows you to create a library of compounds Polymers allows you to generate theoretical polymer sequences Log window allows you to view any error and warning messages When the instrument has been set up and all of the system checks have been made computer control of the instrument can be initiated by starting the MALDI MS program To do this click Chapter 3 Getting started the mouse SELECT button on the taskbar Start button select Programs gt Shimadzu Biotech Launchpad gt MALDI MS Figure 3 3 Windows XP Professional Programs Favorites Documents Settings Search Help and Support Run gt gt b Log Off bill Turn Off Computer Documentation gt H Archiver 3 Compounds Database y 2 Configure Environment Element t Element Database Editor rr Accessories IT Administrative Tools ff Shimadzu Biotech Launchpad T Quadrala Q 5 Enzymes IT RoboHelp Office a pies k Launchpad martDraw ine Soundmax ae o Client
495. tion check file and directory write permissions on the directory being written to and disk space Unable to get file statistics for reference file filename Check that the data files for filename exist in the selected data directory Unable to open reference file filename Check that the reference file filename exists in the selected ref directory Error reading from reference file filename occurred while operation Retry loading filename the file may have been corrupted or may contain invalid information There are no coefficients in the filename file Use a new calibration valid for the mass range of the loaded dataset This file may be corrupt Cannot allocate memory for combining mass time lists Close other programs shutdown active processes to free more memory Memory allocation failure size value Close other programs shutdown active processes to free more memory If value is ve or very large then the parameter set may be corrupted Delete the selected parameter set and restart MALDI MS Error 5000 Error 5010 Error 5020 Error 5030 Error 6000 Error 6010 Error 8010 Error 8020 Error 8030 Error 9000 Error 9010 Chapter 44 Summary of error messages Unable to load comments file Check that the comments file exists in the selected comments directory Unable to open comments file for reading Check that the comments file exists in the selected comments directory Error reading fi
496. tion contained herein is confidential and the property of Kratos Analytical Limited and is supplied without liability for errors or omissions No part may be reproduced disclosed or used except as authorised by contract or other written permission The copyright and the foregoing restriction on reproduction and use extend to all media in which the information may be embodied Trademarks Axima Launchpad and QIT are trademarks of Kratos Analytical Limited Matrix Science and Mascot are a copyright of Matrix Science Ltd Sigma is a trademark of Sigma Aldrich Inc Windows Windows XP and Windows Vista are trademarks of Microsoft Inc Accuspot and Probot are trademarks of Shimadzu Corporation The Mascot Parser supplied with Launchpad includes software developed by Apache Software Foundation Xerces C XML Parser e Jean loup Gailly and Mark Adler zlib e University of California Berkeley and its contributors Regex Library About this User guide Chapter 1 Introduction Printed guides Getting started guide The Getting started guide provides you with an introduction to the features that you will need to use to perform an experiment This book is supplied with the Axima Customer support guide The Customer support guide contains various sections about the Axima including starting and stopping the Axima changing the desiccant etc This booklet is supplied with the Axima Target plate user guide The
497. tions involved in using the Microsoft Windows operating system The PC and the Axima should both be plugged in to the mains supply Switch on the Axima instrument first and then the PC the PC will boot up to the Windows desktop any desktop icons and short cuts will be displayed along with the Launchpad window You can also start Launchpad from the Start button as shown in Figure 3 1 a fm Programs H Archiver 3 Compounds Database w Favorites gt 5 3 Configure Environment IT Accessories 5 7 amp Element Database A Documents 2 ES mos ae Administrative Tools fs Element Database Editor 2 Settings raga Shimadzu Biotech Launchpad k IT Quadralay amp 3 Log Window 2 Search RoboHelp Office gt FJ mavor ms Help and Support SEB t amp Polymers IT Soundmax A R Sample Plate Editor iol Cas arin i g Search i i bx A a IT Symantec Client Security EA A o hi y 4 z o gt Enzymes Database F Documentation Figure 3 1 The Desktop Throughout this manual the use of the mouse will be indicated by the following actions SELECT indicates pressing the select button left button ADJ UST the adjust button middle wheel and MENU the menu button right button The term double click indicates two presses in rapid succession of the mouse button and click indicates a single press of the mouse button Chapter 3 Getting started This convention is used for a right hande
498. tle string gt lt Database string gt lt Taxonomy string gt lt Enzyme string gt lt MissedCleavages unsigned integer gt lt FixedModification string gt lt VariableModification string gt lt ProteinMass unsigned integer gt kDa lt PeptideTolerance float gt lt PeptideTolUnits enumeration gt Da mDa ppm percent lt MSMSTolerance float gt lt MSMSTolUnits enumeration gt Da mDa lt MassValues enumeration gt Monoisotopic Average lt ICAT enumeration gt true false lt PeptideCharge string gt Valid values are 1 2 3 4 5 6 lt Overview enumeration gt true false lt Instrument string gt Valid values are default MALDI_TOF_PSD MALDI_TOF_TOF lt ReportLength unsigned integer gt Number of entries to ASCII Text Method file format 219 220 Chapter 15 Automated operation report lt MaxSearchPeaks unsigned integer gt Number of peaks to search Multiple FixedModification and VariableModification values are allowed Block lt MS1 gt Applications gt lt OligoAnalysis gt lt ModificationEntry gt lt ModificationType enumeration gt Loss Gain lt ModificationMass float mass Da gt lt ModificationMaxPercent unsigned integer gt 0 100 percent lt ModificationEntry gt lt MinLossDivisor unsigned integer gt minimum loss checked parent minlossdivisor lt MaxGainMultiplier unsigned integer gt maximum gain checked parent maxgai
499. tput 5 Wi Print C terminus Hydroxy HO gt Fragmentation Sinay charged 7 Oe Export Cation Proton H x Digest V 1d Report a lon Finder Data Storage Get Manual Settings Save Method Load Method Figure 15 17 Method Editor Compare Sequence The Compare Sequence window essentially contains two sections In the Compare Sequence Options section parameters for the peak matching can be set In the Sequence section peptide sequence information can be inputted in a similar manner to the Peptide Calculator see Sequence Calculator on page 601 Method Editor 183 Chapter 15 Automated operation Further sequence settings are provided via the Compare Sequence Settings window accessible via the Settings button as shown in Figure 15 18 below Compare Sequence Settings Load Sequence Protecting Groups Fragmentation Database Files Shimadeu Biotech Launchpad Peptides standards ksq Select Listed 8 out of 8 matches human angiotensin I human angiotensin II bovine insulin A chain bovine insulin B chain poly P14R bovine insulin B chain oxidised Bradykinin 1 7 human ACTH 18 39 Figure 15 18 Compare Sequence Settings window Table 15 1 Compare Sequence Settings tabbed options Load Sequence Protecting Groups Fragmentation Method Editor Loads a sequence from a database into the Compare Sequence panel see Sequence Calculator on page 601 At
500. traction is shown on the processed trace Figure 16 6 Baseline subtraction Subtracting the baseline Chapter 16 Cleaning up data Adaptive threshold peak detection Adaptive Thresholding overcomes the problem of uneven noise levels throughout a spectrum In extreme cases the noise in one region of the spectrum may be larger than the peaks in another region of the spectrum Adaptive thresholding gets around this problem by changing the peak threshold level to follow the noise in the signal The example below shows high noise levels at the lower mass end of the spectrum while there is less noise at the higher mass end The darker line is the adaptive threshold curve It follows the noise level in the signal successfully identifying the peaks 5383 2 3157 5 100 100 6257 9 Threshold 80 3254 2 60 40 20 5000 10000 15000 20000 3200 3300 Figure 16 7 Adaptive threshold subtraction The adaptive threshold curve is based upon the baseline of the spectrum You do not have to use baseline subtraction in order to use adaptive thresholding although the two are connected To choose the adaptive threshold method from the peak detection window click the adaptive threshold radio button lt There are two parameters associated with the adaptive threshold The first is the threshold offset This parameter represents how high above the zero level the adaptive threshold sits This parameter is the same for the conventional const
501. ts the most abundant naturally occurring isotopes As the number of atoms increases the individual isotopes are no longer resolved as can be seen from the following examples showing 10 15 20 and 25 tin atoms at 2000 resolution Displaying isotopic distributions Chapter 23 Displaying simulated data Molecular formula SatO Resolution 2000 at 50 Molecular formula SatS Resolution 2000 at 50 int Wint 100 100 a a wo a a 2 pal 0 PA al oj 1170 m5 1160 1165 1190 1195 1200 1205 1765 1770 1775 1780 1785 1790 1795 1800 MaseiCharge MasaiCharge Molecular formula Sn20 Resstution 2000 at 50 fomula Sn25 Resolution 2000 at 50 Kim im 100 100 2564 50 w 6 60 6 40 40 a 2 0 0 255 20 2565 270 275 m 2985 MasaiChaege Mace Charge Figure 23 3 Merging isotopes at high mass Displaying isotopic distributions 2419 Chapter 23 Displaying simulated data Theoretical spectra for peptides The simulated data functionality of MALDI MS can be extended to predict spectra for peptide fragmentation patterns comparison of such simulated spectra can be compared with actual spectra to aid in confirming or eliminating postulated peptide sequences At high mass the individual isotopes are no longer visible and all that is seen is the envelope of the isotopic distribution Figure 23 4 shows Trypsin a more complex molecule C1012H1600N2820321S14 simulated at a resolution of 200 as may be obtained in linear
502. ttons position the mouse pointer over the required button and click SELECT J angio2_msms0005 MALDI MS of x File Edit Yiew Instrument Processing Help 7 alal y zel aj al x 04 I_ Display Spectrum gt Profiles 1 54 Masses 176 1023 ieil Data angio2_msms0005 4 Mar 2005 14 37 Cal tof 4 Mar 2005 14 37 CID of 1046 51 3 Shimadzu Biotech Kompact MALDI 6 v2 7 0 Build 20050524 Mode reflectron_ms_ms Power 73 Gate 1035 00 1060 00 P Ext 1050 bin 4 lnt 7 2 mV Profile 54 100 6 6 m sum 356 mv Profiles 1 54 Unsmoothed Baseline 80 255 068 600 MassiCharge For Help press F1 Figure 20 2 Display toolbar Docking the display toolbar The toolbar can be dragged to the left right top and bottom of the display area by clicking on the edge of the toolbar and dragging it to a new position When the outline of the toolbar The Display Toolbar 337 338 Chapter 20 Managing Data Displays changes size over an edge of the window the mouse button is released and the toolbar will dock with the new edge as shown in Figure 20 3 angio2_msms0006 MALDI MS File Edit Yiew Instrument Automation Processing Help eae 2 Bl X E Disple fspecum z P Data angio2_msms0006 G7 c 4 Mar 2005 14 37 Cal tof 4 Mar 2005 14 37 CID of 1046 4 Shimadzu Biotech Kompact MALDI 6 V2 7 0 Beta 1 Mode reflectron_ms_ms Power 73 G OLX lnt 480 mV sum 25928 mV Profiles 1 54 Smooth Av 20 1046
503. ture will calculate the two thresholds using only peaks within that segment Mass ranges set to 3 4 3p 6 2 ELERT A T ll ak T 250 300 350 400 450 500 550 600 650 700 750 800 850 900 MYI Double threshold Double threshold Double threshold calculates calculates calculates thresholds using thresholds using thresholds using these peaks these peaks these peaks The overall mass range is determined by the range set within Peak Picking Double threshold 255 256 Chapter 16 Cleaning up data Peak picking Peak picking Peptide mass fingerprinting is the technique normally used in rapid identification of the protein and the monoisotopic mass is commonly the only peak used in this process Axima instruments are of sufficient resolution to allow the spectrum of a peptide to be resolved at isotopic peak level the monoisotopic peak is at the lowest mass containing only the isotopes C12 N14 O16 and S32 The Monoisotopic picking tab of the Peak Cleanup window has a facility for detecting only monoisotopic peaks The underlying algorithm initially uses the normal smoothing and baseline subtraction functionality of the peak cleanup window but then follows the method of Breen et al reference 1 using a Poisson model to identify the isotopic peaks and thus select the monoisotopic masses The method can also be successfully extended to deal with overlapping distributions of isotopically resolved peaks
504. ty E x 9 peaks calculated Figure 40 2 Polymers window Generating a polymer series Type in the formula for the polymer into the Formula entry As with the Compounds Database and Search windows this can be any elemental or compound formula see Rules for entering formulae on page 573 In the case of polymers the repeating monomer should be enclosed within parenthesis and followed by n to signify that this is the repeating unit For example Formula CH3 CH2 n CH3 H NH CH2 5 CO n H Next type in the Average molecular mass of the polymeric material being generated This is the average polymer weight which the software will use to generate a list of polymer masses around this average weight Type in a Mass window which will limit the range of the calculations to the width of the window about the average molecular mass Type in a value for Minimum Introduction 591 592 Chapter 40 Polymer simulation Reference nylon6 Int 100 80 intensity The generated polymer series will have this intensity at the start and end of the series with a Gaussian distribution in between Average molecular mass 2000 Da Resolution 2000 at 50 2038 12 i 1925 45 Mass window 1000 Da a 2151 28 2264 76 1699 30 Figure 40 3 Generated polymer reference for Nylon6 The table generated can be based upon average most abundant or monoisotopic masses this is selected using
505. u incorrectly typed p r for example this error will be displayed Table 34 7 Errors Using the command line editor 553 554 Chapter 34 Batch processor XML export The p switch was used without a filename This error will be displayed when the p is used on its own The I switch can only be used once The program does not support multiple log files The log file name contains invalid characters You cannot specify a path the log file will be created in the output directory The folder permissions in the output directory prevented the log file from being generated A serious operating system failure has occurred and it is recommended that you restart the PC You have entered a switch which has not been recognised For example wrong Table 34 7 Errors 404 No parameter filename specified 501 Only one log file is allowed 502 Invalid log filename specified 503 Unable to open log file 901 Unable to start processing 000 Invalid switch Log File If you used the I switch the software will generate a log file containing a more detailed view of operations For example 2006 10 23 14 18 2006 10 23 14 18 Batch Process started C Data linear test 2004_11_18 ANAG 0002 run Initialising XML engine Generating c temp bill 2004_11_18 ANAG_0002_B1 mzXML Generating c temp dave 2004_11_18 ANAG 0002_C1 mzXML Generating c temp dave 2004_11_18 ANAG_0002_D1 mzXML Generating c temp dave
506. ubject to the minimum width criteria specified in the width parameter Note that this means that a peak that has an apex value greater than the threshold may still not be reported if its width between these threshold values is too small The threshold and the apex in Threshold Apex peak detection should not be confused since the Apex value in this case is the reporting method not the detection method In general the Gradient Peak detection 247 248 Chapter 16 Cleaning up data Peak detection Centroid method is the preferred method since it is less likely to be affected by noise at or around the threshold value specified when using the Threshold Apex method peak eee EAR bacs threshold Figure 16 8 Threshold cut off point Chapter 16 Cleaning up data The threshold value to be used is set on the Peak Cleanup window MpPeak Processing BEI Dataset JE 1 angi1__0001 A Tace fi Sample 021 a Peak Cleanup Peak Picking Peak Filtering W Scenario Advanced 7 Advanced Settings Profile average Allprofiles Tagged profiles Peak width 20 hans th Peak area Oe OF Smoothing method averacee O x Smoothing filter width jo a chans es IV Subtract baseline Baseline filter width 80 a chans t4 Peak detection method Threshold 25 Centroid z Threshold 25 Centroid Peak Detection Settings Double Threshold z JH Threshold type C H ic pitty Threshold offset 0 50
507. uence will cycle back to the first display The display toolbar zoom buttons have a toggle action Press once to enlarge zoom in and a second time to zoom out The only buttons which do not have a toggle action are the unzoom button 28 and the zoom next display button Resizing displays The zoom width button when used in conjunction with the Shift key increases the width of the column containing the selected display by a factor of 11 and the zoom height button likewise increases the height of the row containing the selected display However should this prove insufficient a particular arrangement of displays can be generated by resizing the displays manually Position the cursor close to the display highlight border and the cursor will change from an arrow head into a splitter bar cursor Figure 20 9 the display borders can be moved to the right or left and up and down until the desired size is achieved Multiple displays 347 348 Chapter 20 Managing Data Displays pep_mix_2466d0005 MALDI MS Oy x File Edit view Instrument Automation Processing Help glij AEL A al x Display Spectrum gt Profiles 1 142 Masses 1295 1304 IEA Data pep_mix_2466d0005 110 c 10 Feb 2005 10 6 Cal tof 10 Fel Data pep_mix_2466d0005 110 c 10 Feb 2005 10 06 Cal to Shimadzu Biotech Kompact MALDI 6 V2 7 0 Betal Mode reflectr Shimadzu Biotech Kompact MALDI 6 2 7 0 Beta 1 Mode lnt 1 5 m sum 220 mY Profi
508. uired for data cleanup In addition prior to these basic categories profile tagging can also be used to improve the quality of the data lil Peak Processing Bey Dataset i 1 angi1__0001 7 Trace 7 Sample 021 a Peak Cleanup peak Picking Peak Filtering y Scenario advanced baa Advanced Settings Profile average All profiles Tagged profiles Peak width 20 chans TL Peak area a paN 9o Smoothingmethod Average o yl Smoothing fiter width 20 chans E IV Subtract baseline Baseline filter width eo 4 chans Te Peak detection method Threshold 25 Centroid x Threshold 25 Centroid Peak Detection Settings Double Threshold C dh Threshold type one e Threshold offset oso my StL Threshold response 100 x tf Apply to Tiles hal pe Apply to Samples I o t Advanced e Poisson Figure 16 2 The Peak Cleanup window Introduction 237 238 Chapter 16 Cleaning up data Combining tagged profiles Often the data collected can be improved in a number of ways Firstly any number of the collected profiles may contain nothing but solvent matrix or uninteresting background noise particularly data collected from electrophoresis gels These profiles can be discarded after data have been collected by tagging specific profiles of interest The method of peak tagging is described in Tagging peaks using the Chromatography window on page 511 This feature will only be
509. uires spaces then the delimiter character must be a TAB The name of the data file or its location including drive letter and full path If no data file is specified the experiment will supply a default name ASCII text experiment file formats 21l 212 Al Firstsamp A2 nd sample text C methods meth1 mtd gt Well ID Chapter 15 Automated operation Table 15 9 Delimited Ascii text Experiment file fields Field g i Name Obligatory Description Method No The name of the methodfile or its location including drive letter and full path If none is supplied the experiment will supply the last method file used or entered in this file or else flag the line as having an error The two file fragments examples below are given to illustrate the above rules The first is for a file with a predefined sample plate where no sample spot size or location information is required The first example highlights an example of each of the Field Names in Table 15 9 on page 211 above and a white space delimiting character Two other fields are also recognized these are primer mass and noprint The first of these specifies a mass value and is primarily intended for and is mandatory in the Oligo Analysis experiment see Automated Quality Analysis of Oligomers on page 187 The second field specifies that any general output spectra etc is to be suppressed for the current sample These two fields are not restricted to any pa
510. ument and make sure that all users are aware of the safety aspects and operating parameters of the instrument FCC compliance This equipment has been tested and found to comply with the limits for a Class A digital device pursuant to part 15 of the FCC rules These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment This equipment generates uses and can radiate radio frequency energy and if not installed and used in accordance with the instruction manual may cause harmful interference to radio communications Operation of this equipment in a residential area is likely to cause harmful interference in which the user will be required to correct the interference at his own expense This instrument complies with Canadian ICES 003 Class A EMC requirements This instrument is CE mark compliant for EMC and in accordance with the low voltage directive Electrical supplies A mains supply to the instrument is required which must be earthed and if the computer and printer are to be situated in close proximity to the instrument a further three supply sockets will be required Safety statements and warnings Chapter 2 Safety warnings The colour coding of the mains supply cable will vary depending on the destination country Table 2 1 Wiring colours Europe U S A and Japan Live Brown Live Black Neutral Blue Neutral White Earth
511. ursors indicating sample bin values 360 Cursors Chapter 20 Managing Data Displays Cursor width The moveable range cursors are normally shown with a small cross hair The options are illustrated in Figure 20 19 General Graphs Graph Text Text Report Cursors Peak Labels Type Normal E Pos 1 Width None Normal Display Window Height Graph isplay window Join with Off it lon Gal 4 md E ie sample 1 1767 ms2_0001 MALDI MS OF x File Edit View Instrument Automation Processing Helg tlie 4 53 ja R Difplay Spectrum Profiles 1 108 Masses 1092 1540 gt l a Int Int 100 50 A 50 int 1 0 GInt 1 0 bd 2 x 2 2 wo wo o base wo t oo 4 MW o A gt rf g 2 F y a n 100 z 400 z i i i j a a i hath canned E o 4100 4200 4300 1400 4500 109 0 4100 1200 4300 1400 4500 11 0 m z mZ K E Mass 1376 42 Closest peak M dM 340 46 1311 94 NUM Figure 20 19 Examples of cursor widths Cursors 361 362 Chapter 20 Managing Data Displays The Display and Window options are useful for measuring resolution or finding the intensity of small peaks as the intensity level can be easily read off the intensity axis Cursors are hidden by pressing the a button on the Display toolbar or using the keyboard pressing Ctrl while clicking the mouse ADJ UST button on the display Cursors can be drawn
512. use click on the Selection or Selection Detail plate displays when the pointer is over the required well or by dragging the mouse with the left button down across the wells of interest To drag select wells in the Selection Detail select Select Wells from the detail s menu which is activated by a right mouse button click when over the detail Note that an expanded view of the Selection plate or the Experiment plate is available by selection from the pull right menu on the plate diagram The expanded view has the same Expand experiment plate functionality as the normal view however it can also be used to identify a sample well moving the mouse pointer over the expanded plate view causes the well id under the mouse pointer to be displayed at the bottom of the window The region of detail can be moved by holding down the left mouse button when in the detail area on the Selection plate and dragging to its new position The same can be done with Selection Detail except that Move Wells must be selected from the menu available by holding down the right mouse button over the Detail view Auto Experiment m D h Chapter 15 Automated operation Table 15 6 Auto experiment plate icons Select the type of plate that will be used in this experiment A file open type dialog box is displayed which is used to select one of the plate files that have already been created by the Sample Plate Editor Select Plate 2 x Look in
513. ut directory If left blank output files are placed in the source folder s If the directory includes spaces place quotes around it Is Searches all subdirectories in addition to the current folder or input folder if specified Table 34 2 Input Output folders Processing Parameters p tof params p c p d Use the processing parameters in this file If the file path includes spaces place quotes around it Use the current processing parameters Use the default set of parameters This option will be used if no p switch is specified Table 34 3 Processing parameters Using the command line editor 549 550 Chapter 34 Batch processor XML export Reporting ly Answer yes to questions suppressing non fatal error messages q Quiet mode No textual output during processing Does not suppress fatal errors or warnings requiring user input log file Create a log file in the output directory If no filename is specified the program will default to using the name run2xml log h no switch Show help overriding all other switches Table 34 4 Reporting Example usage This section provides a few examples of how to use the command line switches described above run2xml run mzdata o C Rods Data XML s y Converts all data files in the current directory including all subfolders to mzData format Non fatal error messages will not be displayed the output directory will be cr
514. v Mascot v Output Data Storage Minimum Mass 400 Maximum Mass 1500 Min Peak Intensity p a X Sequence Order Increasing intensity gt Good PMF Score p5 Max Confirmation Peaks po a Max Investigation Peaks 2 sanua i Save Method Load Method Figure 15 31 Method Editor Peak Selection The peak selection window contains a number of parameters the user can set which are used as a criteria for MS MS analysis Peaks selected for MS MS must be above the Minimum mass and below the Maximum mass Peaks must be greater than the Minimum peak intensity of the largest peak intensity in the spectrum Set the order in which the peaks are sorted for MS MS by selecting increasing decreasing mass or increasing decreasing intensity from the Sequence order field When a Mascot PMF search is performed on the parent MS data two sets of MS MS acquisitions are carried out One set of peaks is derived from peaks that had matches in the first MS MS hit the Method Editor 201 202 Chapter 15 Automated operation Method Editor confirmation MS MS and one set from peaks that did not have matches inside the first PMF hit the investigation MS MS The maximum number of peaks selected for confirmation PSDs is set by the Max Confirmation Peaks field and the maximum number of peaks selected for the investigation MS MS is set by the Max Investigation Peaks field Setting either of these fields to
515. ve the above mass list to a file click Save List As perform another MS MS experiment on a different precursor and import the mass list from the first experiment Mascot MS MS searches 311 Chapter 18 Protein peptide analysis using Mascot search engine 1 Click Add Files Add Multiple Parents from Files 27x Lookin Gackt l EK sample 12 1722ms2_0001 run sample thursday p sample 12 2329ms2_0001 run 9 sample sample 12 1326 ms2_0001 run p sample 12 2329ms2_0002 run f sample sample 12 1336 ms2_0001 run sample 12 11812ms2_0001 run sample sample 12 1614 ms2_0001 run p sample 10001 run p sample 4 sample 12 1615 ms2_0001 run p sample 10002 run pS sample gt File name Files of type MALDI Data Files run Cancel Max number of peaks 80 Segments 5 Lower peak Higher peak e limit 2 a limit 98 i Specify mass ranges as a percentage of the parent masses in the files Parent masses are Monoisotopic Average 2 Select the required file You can select multiple files However if you select a large number of files the process can take several minutes In this instance a progress bar is displayed to show you the progress 3 Set the four fields that define which fragment peaks are selected see Mass list on page 308 4 Set whether the precursor parent ion mass is monoisotopic or average Click the required radio button 312 Mascot MS
516. ver reason All valves on the instrument are drawn either open or closed indicating their current state Chapter 11 Checking the instrument status A context menu to change the units of displayed pressure can be accessed via a right mouse click over the instrument status diagram as shown in Figure 11 2 Gauge 1 1 1E 6 Lmbar y Millibar Pascal Torr Figure 11 2 Context menu of Axima instruments status diagram Introduction 93 Chapter 11 Checking the instrument status Axima Assurance instrument status The instrument status diagram gives an overview of the instrument status in real time Instrument Status Vacuum state System pumped Tuning mode Linear Instrument mode Linear v2 Pump Supplies Pressure Valves fb Pulsed Atspeed On OK Open Extraction Source Source Accelerating Poor lev ah bev Bin Off Off Closed Fail Fail Fail Figure 11 3 Axima Assurance status diagram The key at the bottom of the diagram indicates the possible states of the units Axima Assurance instrument status Chapter 11 Checking the instrument status Axima Assurance status diagrams key The items on the diagram and their possible states are described in the table below Table 11 1 Axima Assurance status diagram key Item Status Explanation Laser Off On The laser is shown in the Off colour blue when not ready to fire and in th
517. w Chapter 32 Archiving data Select the file containing the archive to restore and press Open The contents of the archive file will be read and the Archiver window will be updated to show the contents of the archive as in Figure 32 8 gt Archiver F bills_data zip Restore from Restore Help t Refresh Clear selections Restore to drive C hd I From drive C lt M Program Files 4 Shimadzu Biotech Launchpad lt a Data J Bill lub angl1__0001 lub angio 2 with theoretical00C lub angio 2 with theoretical00 luk bill_0001 lluk cal_0001 lub psd_angl1__0001 lub psdeal_0001 L TOF_Ref_Factory gt File information File totals 0 bytes in 0 file Show file information IM Figure 32 8 Restoring from an archive file Select the files and folders to restore by clicking on the required items The Restore to drive option will default to that from which the data was archived but any of the mapped networked drives may be selected from the available drop list When the required items have been selected press Restore to begin restoring the files At any time during the process of restoring files the Cancel button on the progress indicator window can be used to stop the process The locations to which the files will be written depends upon the paths defined in the Configuration Editor for all of the file types see Environment Configuration Editor on page 60 In this manner the original
518. w the laser can be fired by pressing the Fire button If Store profiles has been enabled then pressing the Fire button will cause the Data Files window to be displayed Figure 14 10 Data Files MEG Save in Standard peptide mix x e f amp E EJ Fe pl4r_msms0002 run i9 pi4r_msms0003 run My Recent i erie yiereet00l arun iS pi4r_msms0003_to00001 run Documents angio2_msms0002 run S pt4r_msmsooo4 run D angio2_msms0003 run iS pi4r_msms_no_gas0001 run E BD angio2_msms0004 run Desktop E angio2_msms0005 run D angio2_msms0006 run D gf_msms0001 run ry of _msms0002 run D glufib_msms0001 run mixed_refooot run D pi4r_cal0001 run D p14r_cal0002 run D pi4r_msms0001 run J My Documents SE My Computer kid iia File name l z Places Save as type Data Files run x Cancel Figure 14 10 Data Files window This is a standard file dialogue from which the folder and file name should be selected under which the data to be collected will be stored If required the collected data dataset can be saved to a file bearing the same name but with the latest run number for that data For example selecting filename mydata0004 will use mydata as the dataset name but if 25 runs have already been collected for that dataset name i e runs mydata0001 mydata0025 then the next data collected will be saved as run mydata0026 Firing the laser Instrument Chapter 14 Collecting data from a sam
519. ween x displaying the normal status view of the experiment plate to one showing test results status Pass Fail or Uncertain following Oligo analysis of the samples Select whether or not a view of the Experiment plate is sent to the default printer at the end of the experiment Right click on the icon to select the properties menu controlling header information printed along with the plate diagram see Figure 15 25 on page 193 Allows the user to align the current plate using the same procedure as described in Plate Alignment for Axima instruments on page 128 Experiment Controls The experiment controls are used to open save and run experiments There is a text box directly above the toolbar that is used to display the current status or activity of Auto Experiment Table 15 7 Auto experiment experiment icons a Create a new empty experiment The currently L selected plate type is used as the initial plate for new experiment Select open a previously saved experiment A file open type dialog box is displayed which is used to select a previously saved experiment file The name of the current experiment file is displayed in Auto Experiment s window caption Save an experiment with a user chosen name A file ie save type dialog is displayed which is used to selected the disk drive directory and name of the file The file saved becomes the current experiment file Save the current experiment If
520. wer 75 Gate 1525 00 1540 00 P Ext 1535 bin 64 lnt 2 3 m sum 333 mV Profiles 1 144 Smooth Av 20 Baseline 80 Tolerance cursors 852 03 100 908 17 80 1005 37 60 Mass Charge Mass 949 16 Closest peak M dM 258 84 Figure 20 17 Tolerance band cursors Cursors 359 Chapter 20 Managing Data Displays Time cursors With Type set to Time cursors can be set to report the sample bin value of the point on the graph under the last moved cursor The collected data is made up of sample values of up to 128K 131 072 bins depending on the operating mode and model of instrument By moving the cursor across the graph the sample bin corresponding to the cursor position is reported in the base window status bar Figure 20 18 When both cursors are displayed the sample bin difference between the two cursors is indicated in the base window status bar angio2_msms0006 MALDI MS O x File Edit view Instrument Automation Processing Help gll YE 2j E h2 lt Display Spectrum X Profiles 1 v Masses 313 375 Ie Data angio2_msms0006 G7 c 4 Mar 2005 14 37 Cal tof 4 Mar 2005 14 37 CID of 1046 51 Shimadzu Biotech Kompact MALDI 6 V2 7 0 Beta 1 Mode reflectron_ms_ms Power 73 Gate 1035 00 1060 00 P Ext 1050 bin 53 lnt 3 4 m sum 182 mV Profiles 1 54 Smooth Av 20 343 26 100 80 60 40 335 340 345 350 Mass Charge Sample 47450 Figure 20 18 Time c
521. which were saved previously and merge them with the currently defined labels click on Include the Open window will be displayed Select the labels file to include and press Open Annotation Properties Se x Type Text 4 Data Properties Dataset x Mass from Trace Z to Display Properties Font Arial Bold T Italics J Size jao a x10 Underline I Angle 0 degress Colour E Set Transparent C Opaque Apply Cancel Background Figure 20 46 Annotation Properties windows Annotation 393 394 Chapter 20 Managing Data Displays Annotation Double clicking on a label also displays the Annotation Properties window At the top of this window the Type of the label is indicated If the label comprises user defined text rather than computer calculated i e AM or M AM then the Text box will be editable allowing the text in the label to be changed The Label visible check box indicates whether the label is to be shown or hidden in the current display In the Data Properties area of the window Labels can be applied to any loaded dataset by selecting from the drop down list at the Dataset option A label can be moved from one trace to another by selecting the Trace type on this window The mass position of the labels can be adjusted by changing the Mass from and to where applicable In the Display Properties area of the window the colour and background opacity of the
522. will be loaded Press Load defaults to use the factory defined MALDI MS colour scheme Changing chromatogram trace colours 435 436 Chapter 24 Choosing user defined colour schemes Changing distribution trace colours On a theoretical distribution trace the lines which show the mass distribution of the molecule and its adducts can be shown in user defined colours To change the colours defined for theoretical isotope distribution displays select the Distribution Colour Editor shown in Figure 24 6 W Colour Editor Distribution BEI Traces Molecule Adduct 1 Adduct 2 Adduct 3 Adduct 4 Colour sets Save Load Load defaults Figure 24 6 Colour Editor for isotope distribution traces There are four colour options on a distribution display Molecule and Adduct 1 4 for the overall isotopic distribution of the parent molecule and up to four adducts respectively see Displaying simulated data on page 415 The colours for these traces can be defined by the user in the same way as for the other graph types Having made all of the required selections press the Apply button to apply the selections to all of the displays This will apply the new colour scheme to the displays but the colour scheme modifications will not be saved in any way they are only temporary To save the user defined colour scheme so that it will be made the default colour scheme and loaded automatically when the MALDI MS software
523. xample a calibration has failed but the Automatic program has continued with the next sample Error messages indicate problems that need attention perhaps due to a hardware failure or imminent failure Notification feature This feature allows you to send emails when the Axima software generates an event 1 From the Events Logged window click the Update Email button Recipient Name Email Address Error Warming Information Audit Suc Audit F Service engineer service kratos co uk Vv Vv Vv oO B 2 Enter the form and click the OK button If you wish to direct emails to an off site service engineer or service centre seek their permission first Eventslogged 71 72 Chapter 7 The Log Window Events logged Chapter 8 Loading and unloading data Chapter 8 Loading and unloading data 74 Chapter 8 Loading and unloading data About data on MALDI MS The first menu option on the base window is the File menu which controls all aspects of loading and saving data and parameters Three terms will be used in this section namely data statistical files and parameters e Data refers to any results collected by the instrument from any of the samples It also refers to any statistical information such as the number of shots mode of operation number of shots averaged e Statistical files having a run extension are created automatically when data is stored Parameters are the settings of the v
524. y Storage slide Raster Tuning T Auto quality poser feo M AH eoit oa Profiles o a per sample n Shots 20 v accumulated per profile Ton Gate Da lof on Bank fpo ied I Low Mass Zoom r a po IX Pulsed Extraction optimised at Da C H ioral x suspend Bort lt pasa t ee se aa X a5 Acquiring sample G14 Acquiring profile 1 of 100 Acquisition Maintenance QIT ToF Experiment T Accumulate Profiles to File Instrument Maintenance x Purge CID Gas Optional features and instrument specific items are in red Axima Performance only Figure 5 7 Instrument menu For details of purging the CID gas lines in an Axima Performance see Introduction on page 640 Instrument menu Chapter 5 Window and menu guides QIT Maintenance x fre Jes Tech Auto Qualty Storage Side Raster Tuning QtT ToF m5 F Ago gusty seytt w sid En E CID Gas 1 fArgon Purge Argon Profiles 500 a per sample Bets T sccumdated per profile bel Pe ma FS Purge He Gas eee aox ale a wei D Activote Acoma es Please type the advanced user password Password Acquisition Maintenance QIT ToF Experiment Optional features and instrument specific items are in red Figure 5 8 Instrument menu Axima Resonance Instrument menu 43 44 Chapter 5 Window and menu guides Acquisition tabbed menus This w
525. y by 2 Ctrl Decrease font size by increasing number of lines in the display by 5 Increase font size by reducing number of lines in the display by 5 The Display Toolbar 341 Chapter 20 Managing Data Displays Multiple displays The display area of the window can be split into any number of individual displays When the window is first opened it will show a single display New displays can be added or existing displays removed at any time and each display can show different views of the data By creating more displays several graphs or text reports can be shown at once For example spectra can be shown in one display with a mass list of mass assigned peaks in another display for comparison Figure 20 4 angio2_msms0005 MALDI MS Ol x File Edit View Instrument Automation Processing Help re ESL 2j E x A gt Display Spectrum 7 Profiles 1 54 v Masses 34 1020 Mass Area Total Apex m Resolution S N Flags Mass Area Total 6994 23 57 2 77 6 40 128 04 30 71 U 1045 02 0 11 0 07 70 08 31 81 3 74 6 48 129 16 30 93 U 1046 51 100 00 68 33 71 99 22 70 2 67 3 83 167 28 17 31 U 1046 86 0 38 0 26 86 02 67 67 7 95 3 56 196 39 14 31 1047 50 27 66 18 90 86 93 21 70 2 55 5 88 213 97 24 27 U 1047 89 0 24 0 16 87 13 14 86 175 5 01 193 28 20 56 U 1048 53 11 39 7 78 109 97 100 00 11 75 12 11 280 57 61 90 U 1049 52 403 2 75 110 15 22 49 2 64 8 48 226 28 43 19 U 1050 52 0 92 0 63 112 02 2248 2 64 3 52 2
526. y toolbar see Figure 20 13 above The data displays make extensive use of cursors to mark and delimit individual peaks and ranges of peaks They are used in calibration to assign accurate masses to known calibrant reference peaks They are used with chromatograms to select sweet spot areas on the sample slide for laser aiming A particularly important use of cursors is to display the mass difference between any two points on a graph Figure 20 14 J pep_mix_2466d0005 MALDI MS ojx File Edit View Instrument Automation Processing Help bed db Ei 2 a h lt Display Spectrum v Profiles fi v Masses 1 040 1058 Ie Data pep_mix_2466d0005 11 Ofc 10 Feb 2005 10 06 Cal tof 10 Feb 2005 9 02 Shimadzu Biotech Kompact MALDI 6 2 7 0 Beta 1 Mode reflectron_ms Power 45 Blanked P Ext 2460 bin 91 lnt 0 5 m sum 69 mY Profiles 1 142 Smooth Av 20 Baseline 80 1047 46 100 Click left mouse button once to select meremas Click a second time to select the end mass a The mass difference dM and resolution are reported 1042 1044 1048 1050 1056 108 e Mass Charge Mass 1049 79 Closest peak M dM 2287 29 dM 3 94 MjdM 266 11 T Figure 20 14 Using the mass difference cursors 354 Cursors Chapter 20 Managing Data Displays Range cursors are useful in determining the fragmentation losses within sample spectra There is a Customise cursors window available which provides extra curs
527. yped into the Comments window File gt Comments This is the Prefix line from the Comments window This is the sample spot comment for the sample spot 1 from which this data was collected This is the name of the data as selected in the load window angio2_msms or if collecting data as given in the Choose data window It includes the run number 0001 and the sample spot number after the full stop G7 The folder name bill is displayed using the Display folder field The sample spot number Letter c indicates a charged positive negative spectrum Letter n indicates a neutrals Spectrum This is the date and time on which the data was collected Customising Graphical Reports 375 376 Chapter 20 Managing Data Displays Table 20 5 Display header information Continued Example item Cal tof 4 Mar 2005 14 37 CID of 1046 51 Shimadzu Biotech Axima ToF V2 7 Mode reflectron_ms_ ms Power 73 Gate 1035 00 1060 00 P Ext 1050 bin 53 Description This is either the name of the calibration as selected on the Calibrate window or the method of calibration in this case using the factory calibration tof Collision gas was used The mass 1046 51 is the Parent mass set in the Calibration window The instrument model which was used to collect the data The software revision number Data was collected using the reflectron_ms_ms the alternative would b

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