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flexAnalysis 3.0 User Manual

1. Figure 21 General Mass Spectrum Calibration D dataspepmiss 0 ARA 2006 06 20 10 37 25 UTE ANBTOFOS TOF User DO Methods flexControlMethods AP_Pepmia 25k 3077 par pepmissi Aad perme 20 06 2006 Contents of the General tab Graphical User Interface GUI The General tab Figure 21 is used to display general information regarding the selected analysis such as file name sample mass spectrometer title and comments specific to the current analysis Changes of title and comments can be performed and accepted by pressing Apply pepmix 0_A24 3 Analysis Properties General Mass Spectrum Calibration Group Parameter AcgD ate Acgtethod Comment Comment Comph ame Digitizer Type FLV er InetiD lnstT ype Las lter MoShots OpiD Acquisition Misc Acquisition Autoecute Acquisition Detection Value Acquisition Misc Acquisition Setup Acquisition Spectrometer Acquisition Spectrum Acquisition Target Processing Processing Auto ecute Processing Calibration Processing Misc 006 06 20T 10 37 25 000 D MethodsflexControlMetho pepmis 20 06 2006 ANCTOFOS Scqiis DP240 lesControl 3 0 77 0 35002 00001 ultrafles TOF TOF Laser beam attenuation FB Number of shots 600 Operator ID or name TOF User Figure 22 Contents of the Mass Spectrum tab flexAnalysis 3 0 User M
2. A 2 1 WHAT IS HPC HPC is a fine correction of the calibration in the mass range between 700 and 4000 Da available on ultraflex mass spectrometers The use of any kind of pulsed ion extraction in time of flight mass spectrometry distorts the linear relationship between m z and the square of the ion flight time By empirically fitting the residual deviation few ppm from the ideal quadratic calibration by a high order polynomial HPC one can significantly increase mass accuracy esp for masses below 1kDa For details please refer to Gobom J et al Anal Chem 2002 74 3915 3923 A 2 2 HOW CAN I USE HPC ON THE ULTRAFLEX ultraflex instruments production 4 03 or later are equipped with a dedicated HPC flexControl acquisition method called Proteomics HPC par This method preset at the factory contains all relevant HPC parameters and must not be changed If you change any of the instrument voltages or the deflection pulser settings the HPC method becomes invalid Use Proteomics HPC par for all standard proteomics applications and the HPC correction is automatically performed in flexControl2 x and flexAnalysis2 x without any user interaction for all spectra acquired with this method provided that HPC is not globally switched off in flexAnalysis Use the common calibration and mass list generation procedures for those spectra Note HPC does only eliminate the deviations from the quadratic calibration function and it does
3. YValues 0 El Graphic esport DP 300 Figure 101 Option to specify the numerical precision of the coordinates The Export feature allows the operator to adjust the numerical precision of the x mass and y intensity value Choose these values as small as possible in order to save disk space and processing time The values chosen here are the number of digits used for each data point when exporting a mass spectrum You can define with which resolution your graphics are exported by adjusting the DPI value 2 98 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI 2 4 10 10 4 Calibration Options lid Display ll Labels l Export Calibration Tolerance values used for recalibration Near Neighbor Calibrant 108 ppm Default Calibrant 400 4 ppm High Precision Calibration HPC Apply HPC if available for analysis Figure 102 Option to specify the tolerance of the calibration strategy Settings on this tab are relevant for internal recalibration section 2 4 5 2 The Near Neighbor tolerance applies to the Interactive calibration whereas Default Calibrant is used for Statistical Peptide Also the global use of High Precision Calibration HPC can be activated in this dialog HPC is a fine correction of the calibration in the mass range between 700 and 4000 Da refer to section A 2 for details flexAnalysis 3 0 User Manual Version 3 0 2 99 Graphical User Interface GUI Br
4. E Edit Building Blocks command Annotation menu 2 48 Edit command 2 24 Edit Parameters command 2 53 Edit Script command 2 67 Exit command 2 16 Export command 2 11 F Files for recalibration 2 1 Filter Background Peaks command 2 27 2 29 Find command 2 23 H Help Topics command 2 107 High Precision Calibration 2 99 A III Horizontal Auto Scaling command 2 75 Installation 1 4 Interactive modes 2 108 Internal Calibrate command 2 36 Internal calibration procedure A I L License command 2 104 2 105 Lock all Applications command 2 104 Index Macro Editor command Mass List command Mass List Window Mass Spectrum Command Mass Spectrum Window Menu Bar Microsoft NET Framework O Open command Operator command Options command P Paste Segment Spectra command Paste Special command Poly Tools command Print Preview command Process Spectra command ProteinScape command Purpose of the software R Range Selection command Redo Zooming command Report Designer command Rules for statistical calibration Run Script command 2 94 2 102 2 108 2 101 2 108 2 3 1 1 2 50 2 104 2 97 2 70 2 19 2 82 2 13 2 34 2 82 1 1 Bruker Daltonik GmbH S Save All command 2 9 Save As command 2 9 2 52 Save command 2 52 Scale to Entire Spectrum command 2 74 Select All command 2 20 Select Default command 2 68 Select None command 2 20 Set Display Range command 2 75 Smooth Mass spectrum command 2 32 S
5. fid file on the display windows of flexAnalysis In case of loading FAST spectra that have been acquired with flexControl 1 x and therefore have no valid calibration the dialog shown in Figure 13 appears to load the matching calibration file psd flexAnalysis 3 0 User Manual Version 3 0 2 7 Graphical User Interface GUI Bruker Daltonik GmbH Warning Calibration file not found Please select a valid calibration file Browse Cancel Figure 13 Searching for FAST calibration files 2 4 1 4 Close Files are closed with the CLose command the corresponding icon a of the Toolbar or the short cut Ctrl F4 The feature closes only those analyses that are marked in the Analysis List Window In case the analyses are currently not saved a confirmation dialog appears and asks for saving before closing 2 4 1 5 Close All The Close All command closes all open analyses In case any spectrum is currently not saved a confirmation dialog appears and asks for saving before closing 2 4 1 6 Load and Unload The Load command loads the data of an analysis that is open but not yet loaded Figure 14 in the flexAnalysis treeview from the hard drive in the memory The Unload command unloads them again Alternatively you can click with the mouse in the corresponding checkboxes in the Analysis List Window to load the analysis 2 8 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface
6. Full Hame The selection in the Abbreviation Box for Building Blocks determines whether the Symbol Abbreviation or the Full Name of the currently activated Building Block is shown in the display and in the Annotation Text field An example is shown in Figure 54 flexAnalysis 3 0 User Manual Version 3 0 2 45 Graphical User Interface GUI 517 176 619 261 bs C 0 92 633 006 646 67 577 a4 00 550 BOD 650 517 176 619 261 Cys 0 92 533 886 648 67 Bruker Daltonik GmbH 917 176 619 261 Cysteine 0 92 533 506 646 670 y 577 Ba 00 550 apg 650 Figure 54 Example of different annotations texts Aral lg Center Horizontal ict JA Contr Won The font and font size as well as the position and orientation of the annotations can be determined with these features The color of the Annotation label can be changed via the Tool menu entry Colors 2 4 6 3 Clear The Clear command and the corresponding icon rit of the toolbar are used to delete all Annotations from the spectrum display as well as the possibly resulting sequences from the Sequence list 2 4 6 4 Show Mass Difference The Show Mass Difference command and the corresponding icon TT of the toolbar are used to show mass differences additionally to the respective Annotation name symbol or abbreviation in the display This value shows the mass difference between an assigned building block and the actual mass diff
7. 15 Audit trails are useful to trace back the operations of a user he made in a specific moment The Bruker Compass Security Pack and user management are described in a separate manual flexAnalysis 3 0 provides the following key features e Multiple spectrum display for convenient spectra comparison and analysis e Highly sophisticated algorithms for authentic automated and interactive peak detection SNAP Centroid and Sum Peak finder e Highly sophisticated algorithms for spectrum processing smoothing baseline subtraction e Highly sophisticated algorithms for automated and interactive recalibration of spectra linear quadratic statistical and High Precision Calibration support e MS MS support Presentation and processing of LIFT spectra as well as generation of FAST spectra e Annotation e Spectrum and graphic export e Report designer for creating user defined analysis reports and specific report layouts e Integrated Sax Basic Development Environment for the design of flexAnalysis methods and scripts Sax Basic is Visual Basic for applications compatible e Proteineer Support Integrated data exchange with BioTools and ProteinScape program packages e Data processed in flexAnalysis 3 0 can be delivered in form of spectra and peak lists to further interactive data processing software packages such as GenoTools SNP Single Nucleotide Polymorphisms genotyping analysis e Polytools monomer determinati
8. Enter a file name into the field File name and press the Save button flexAnalysis creates the corresponding file flexAnalysis 3 0 User Manual Version 3 0 2 11 Graphical User Interface GUI Bruker Daltonik GmbH On selecting the Export Graphic command the Export Graphic dialog appears to specify the file name and location to the export a graphic The Export Mass List to Excel command exports the peaklist to Excel An example is shown in Figure 18 This macro exports all available peaklist parameter It is not possible to select only some of them Fd Microsoft Excel Book File Edit Insert Format Tool Data Window Help A SF o mw ME 121 0 ee VIEW Deh st 8 Slav amp 8 KIE x f Intens al Quality Fa Res Area Rel Intens FWHM Chrz Bk Peak 6 1296 463 Figure 18 2 4 1 13 4250 05 39907 26 100910 30060 49 0103 56 167 1143 06 31533 192 5352 73 40560 153 4619 2070 679 4103 61 4452 335 1039 306 1545 655 3046 291 6043 754 4752 095 5AA 513 5149 025 15115 95 1264 41 62996 03 10757 22 44199 04 Mass List exported to Excel Print Preview 0 4603 25 0 395456 0 999955 0 37716 0 773956 0 15678 4663261 0 165551 0 270007 3660404 0 200552 4927130 0 251700 2251640 1857311 FALSE FALSE FALSE FALSE FALSE The Print Preview command and the corresponding feature button ey of the Toolbar open the dialog shown in Figure 19 for further adjustments The prin
9. Spectrum Spectrum 2 Distance i Calculate 1296 655 1306 51 4 3 829 0 097 Calcule 1641 814 1651 826 10 011 1657 810 1667 822 10 012 Saveds Figure 38 AutoDistance dialog e Distance Mass distance value to be searched for e Tolerance The search tolerance It can be specified in Da or ppm e Spectrum First Spectrum The drop down list contains up to two spectra In case of two define the one that is used at first e Calculate Starts to calculate and search for the selected distance e Result List containing the calculations of flexAnalysis e Save As This button opens the dialog to save the result as a CSV file that different applications e g Excel can easily import An example is shown in Figure 39 2 30 flexAnalysis 3 0 User Manual Version 3 0 View Insert Bruker Daltonik GmbH Fd Microsoft Excel distancelist cs File Edit Deh ese Gar 4 Format Tools Graphical User Interface GUI Data Window Help spectrum Spectrum Distance AreaRatio 526 337 566 039 41 702 30 57 664375 566 039 96 336 25 743 664375 713 260 48 913 26 635 664375 739 26 74605 40 498 664375 75 2606 92 0691 225 418 F541 715 2060 44 122 5 805 TREN 739 26 18 15 8 027 Figure 39 Example of a Distance in mass list result 2 4 4 Process Menu Process Calibrate Annotation Method FAST lids Smooth Mass spectrum alla Subtract Mass Spectrum Baseline Undo All Processing Batch Process Process Spectra
10. 2 4 7 6 Ed Rata meters unas 2 53 2 4 7 6 1 POCOS S oi ita 2 53 2 4 7 6 2 FAST only for FAST Segment Spectra ccccooocccccccccnccnoccnnononcnnnnnas 2 63 2 4 7 6 3 Recalibration Masses only for MS Spectra oooocccccoccoccconcnncnno 2 64 2 4 7 6 4 MaS SLIS EELA OU rtccctet atin A ue etaacusay ancemiantaner saan 2 66 ZA7 6 3 DISDIAV science pele iiee iene in eee ie Minder 2 66 2 4 7 7 al Gens Mrmr eet eee ee dad 2 67 2 4 7 8 Select 2 FD ene ene gee eee Rare eee ee eee 2 68 ZAS FAST Mens a a 2 69 2 4 8 1 OVEIE Wii ad id 2 69 2 4 8 2 Paste Segment Spectra vaina ds 2 70 2 4 8 3 Change Parameters cant dnd 2 71 2 4 8 4 Delete Segment Spelt aisadament a E a 2 72 249 NOW Me oi 2 73 2 4 9 1 A 8 et ht cates g cases E dee ed aT ices 2 13 2 4 9 2 AUTO SCAlNG dota 2 74 2 4 9 3 Scale to Entire SPecirUMi ii o a 2 74 2 4 9 4 Horizontal and Vertical Auto Scaling oooccococoncnnoconcnnconncnnononennnonos 2 75 2 4 9 5 Set Display RANG Ouanna ich ealee a a hae 2 75 2 4 9 6 LON e dida 2 75 2 4 9 7 LOOM Dass oes hadnt ses redex tawaseteectesdandeasee act eee desc aes tease anc oteyadanteunteeeoccuases 2 6 2 4 9 8 Undo ZOOMING eaan id 2 76 2 4 9 9 Redo O ONIN Oasan a a 2 76 A O A 2 17 ZAS AS tn odo 2 78 flexAnalysis 3 0 User Manual Version 3 0 V Bruker Daltonik GmbH ZA TOO ME ate a de el e o lo 2 78 ZATOT OVEMIEW aruana a a a a aaa 2 78 2 4 10 2 Report Designer cooncccoccccconnnccconoccnonocnonoconnnnon
11. 4 12 8 Audit Trail Viewer The Audit Trail Viewer command opens the Bruker Daltonics AuditTrailViewer dialog It is only available if the Bruker Usermanagement is installed A Bruker Daltonics AuditTrailViewer UserManagement Audit Fie Open Audit Trail Help Open Audit Trait Electronic Record User anagemernt System Stabe uct trail ig ok Filter All entries y Entries 1824 Filtered Entries 824 E entries Old State Program admin Login Unlocked Unlocked Login Login Login Login Used Login Login on PC 10 45 Application Application on PC 10 49 on PC 10 49 on PC 10 49 on PC 10 49 global right on PC 10 49 on PC 10 49 Locked Locked admin Unlocked Unlocked admin admin admin admin admin admin BOALService 4 2520 BOALService 4 2 52 0 BOALService 4 2 52 0 BOALService 4 2 52 0 BOALService 4 2 52 0 BOL Service 4 2520 BDALService 4 2520 BO ALService 4 2520 BOALService 4 2 52 0 BD ALS ervice 4 2 52 0 DateTime 7 3 2006 3 13 05 PM E 2322006 2 50 04 PM 22322006 11 25 53 AM 672972006 12 17 06 62232006 14 04 13 672972006 11 21 52 672972006 10 34 14 672072006 3 01 22 AM 62202006 3 01 14 AM 6 20 2006 7 53 10 AM Login on PC 10 453 admin BDALService 4 2 52 0 64282006 7 50 56 AM sy BDALSevice4 2520 8 9 7 3 2006 3 13 25 Ph 0 ISO Date Time 2006 07 03715 1325 01 00 iY y iY iY dy de Je Je Je Je amp Type Information Oper
12. 682 46 1002 573 61 6 3532 416 78 1028 578 30 3 3911 209 36 1046 558 55 3599 5472 OF 1251 659 35 5 4511 301 06 1296 701 207 0 4307 1901 16 1332 666 15 3 3429 150 79 e 1347 742 erla 4113 6957 25 pepmi10_4511 1363 72 97 0 4654 1020 18 x 1620 9 1359 742 150 5156 166 80 y 6254 00 E ay 1623023 571 00 E LJ L 1651 794 214 4899 315 50 1667 775 138 4828 184 58 2093046 21 1 6252 13361 3421 152 1315 5797 436 20 2447142 81 4 8220 235 53 2465 153 1470 0 8136 3924 67 3063 234 EB 1523 5 04 3071 449 24 8 11392 28 24 3083 403 179 536 19 18 1620 1621 1622 1623 mlz 3147 500 522 5 9165 786 72 Figure 31 Edit Mass List directly A peak is added to the peaklist but only the columns for Mass Intensity and Relative Intensity are filled because the peak annotation has not been created with a peak detection algorithm that would calculate the other parameter 2 4 3 5 Clear The Clear command the corresponding button Se of the Toolbar and the short cut Shift F5 are used to clear the current mass list of the selected spectrum or spectra Select the spectrum spectra in the Analysis List Window if necessary with simultaneously pressed lt Ctrl gt key whose mass list s shall be cleared click JE or from the Mass List menu select Clear 2 4 3 6 MS MS List The MS MS List command works in connection with the Add to MS MS List comman
13. Bruker Daltonik GmbH Automatic Assign Peaks from the peaklist are automatically assigned to the reference masses a new fit result and errors are calculated but not yet applied to the spectrum If no peaklist is available this is automatically created before the assignment The peak finder algorithm used for automatic calculation is the one currently selected in the processing parameter editor section 2 4 7 6 1 Preview With a click on this button the new fit result calculated with manual assignment or the button Automatic Assign is applied to the spectrum it is moved in the display Undo After the new fit result has been applied with Preview it can be cancelled with the Undo button The original calibration currently saved in the spectrum is applied again and peaks are re annotated Clear Assigned This button deletes the assignments from the calibration list it does not touch the possibly done calibration T After a calibration has been done the dialog can be closed with these buttons OK means Preview and Close so even if the Preview button has not been applied the calibration is accepted Cancel means Undo and Close i e even if the calibration has been applied with Preview before it is not accepted For manual assignment of masses to reference masses it is necessary to click in the spectrum left to the peak of interest Therefore it is helpful to zoom automatic
14. Building Block is always located in the second row Figure 66 Fragment Peak Width This parameter is used to confine a peak to a particular width thus excluding broader peaks from the peak list SILE Mass Difference s H L Enter one or more values separated by a semicolon to define the mass difference between the Heavy H and Light L isotopes of the labeling reagent The SILE mass difference s defines the m z distance between the two or more labelled forms E g for 18 0 digest labeling it would be one and two times m z 18 0 m z 160 2 004 and 4 008 2 58 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI The knowledge about the SILE mass differences is used for an improved S N thresholding and the internal judgment of the presents of overlapping monoisotopic peaks SILE Partner SN Threshold The SILE Partner S N Threshold allows the definition of a second S N threshold which is checked for SILE partners If there are two SILE partners only one has to match the normal threshold For the second partner it is enough if it is large enough to match the SILE partner SN threshold This gives a better dynamic range for the observable regulation without adding too many noisy peaks Peak detection algorithm Sum The Sum peak finder Figure 67 uses a time saving approach while calculating so called pseudo derivatives instead of real derivatives as known from Centroid If
15. Graphical User Interface GUI Bruker Daltonik GmbH 2 4 1 10 Sign Electronic Record The Sign Electronic Record command is used to sign spectra electronically when they have been processed It is only available if additionally the Bruker Compass Security Pack is installed After a spectrum is signed it is write protected To sign a spectrum it is necessary that the user who wants to sign has the Usermanagement given right to sign This is checked during the sign process when the user fills in his Operator name and password Figure 15 The field Meaning may be used for comments Sign Electronic Record Operator Password M eaning Figure 15 Sign Electronic Record dialog 2 4 1 11 Show Signatures The Show Signatures command is also only available if additionally the Bruker Usermanagement is installed and becomes active if a signed file is marked in the treeview It opens the View Signatures dialog MM View Signatures D IDATA WemoWBiotoolsiHlexiBatchDataWwarp2_discoverW_G1511115Ref E Date Signed by Meaning Valid 006 07 04771 5 11 03 968 01 00 John Smith PH has checked unknown Check Validity Figure 16 View Signatures dialog 2 10 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI e Date This column shows the date and your local time The value most right in this example 1 00 corresponds to GMT your time zone e Signed by This column contains t
16. Help Menu in the flexAnalysis Macro Editor for a detailed description on how to open to create and to execute macros within the macro editor 2 94 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI 2 4 10 9 Customize The Customize command is used to vary the contents of the menu bar Additionally the user can remove or add parts of the toolbar These features are available on the tabs Toolbars and Command of the dialog Customize Customize Toolbars Command Toolbars Le Show Tooltips New File Cool Look z Reset Large Buttons Figure 96 Toolbars tab displayed by default After opening this dialog shows first the Toolbars tab which provides default palettes that contain feature buttons to be temporarily removed or docked on to the GUI by applying the corresponding checkbox there is no access to the checkbox Menu Bar The button New opens the New Toolbar dialog in order to specify a name for the new empty palette that will appear at once on the Graphical User Interface after closing with the OK button Hew Toolbar Toolbar name o DK enter a name Figure 97 Creating a new Toolbar name flexAnalysis 3 0 User Manual Version 3 0 2 95 Graphical User Interface GUI Bruker Daltonik GmbH The symbols of the Command tab Figure 98 may be used to add one or more functions to the new palette For instance
17. Right click the selection and select Paste or Paste Special or Click E 2 4 7 3 Save The Save command or the corresponding icon _ of the toolbar are used to save changed method settings to the same file name This overwrites the original not write protected method If the opened method has been changed but not saved it is shown by the modified indicator a symbol which is shown in the title bar of the Method Parameters Dialog box or in the Script Editor If a modified method is not saved the current parameter settings for an analysis will be saved when this analysis is saved The file Analysis FAmethod is created in the analysis tree Selecting the Save command removes the modified indicator previously appended to the title bar of the method editor Note lf the original method is write protected the Save command is not accessible and the Save As dialog opens 2 4 7 4 Save As The Save As command is used to create a new processing method by saving a changed method to a new or already existing file name Enter a new or select an existing processing method name in the appearing dialog The new file name will be displayed in the status bar at the bottom of the GUI and in the title bar of the corresponding Method Parameters Dialog box instead of the previous method name 2 4 7 5 Run Script The Run Script command the corresponding icon gt of the Toolbar and the short cut F2 are used to run the Sax Basic script that be
18. Zoom Range of M O From Mass Control List Calibration Fit Order O Near Neighbor Quadratic User Defined A 500 ppm B mE Figure 73 Method parameters dialog box Recalibration Masses The parameters selected and adjusted in the Recalibration Masses tab determine the results of the internal calibration The settings correspond to the settings in the Internal Calibration dialog section 2 4 5 2 The Recalibration Masses tab is used in context with methods and automation the Internal Calibration is used for manual calibration Select first the calibration strategy you want to use The adjustable parameters on this tab have been explained in detail in section 2 4 5 2 The pull down menu Mass control list contains predefined mass control lists that can be used for recalibration The lists can be modified with the Edit button The Mass Control List Editor opens and shows the selected list Figure 74 2 64 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI PeptideCalibStandard mong Peak label m_z Tolerance pp Lalibrarit Background Adduct Calibrant frequency d Bradykinin 1 7 757 399160 50 al Angiotenzin Il 1046 541800 al Angiotensin 1296 684800 AD Substance P_ 1347 735400 50 Bombezin M H 1619 822300 al Renin Substrat 1758 932610 AD ACTH_clip 1 1 2093 056200 50 ACTH _clp 18 3 2465 198300 50 Somatostatin 314747000 Al ai lt i 4 al 4 lt i al al lt l E
19. a spectrum to the total spectrum This command has the same effect as Auto Scaling however the Auto scale mode is not switched on after this command is executed 2 74 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI 2 4 9 4 Horizontal and Vertical Auto Scaling The Horizontal Vertical Auto Scaling command and the corresponding feature button V are used to display the entire mass intensity spectrum in the Mass Spectrum Window 2 4 9 5 Set Display Range The Set Display command is used to specify ranges for abscissa and The Set Display Range dialog box opens which allows setting the mass range and the intensity scale when the checkboxes Auto Scaling are not checked Set Display Range Or Mass m z 838 1070 Auto scaling Intensity 105 e Auto scaling Figure 85 Set Display Range dialog box 2 4 9 6 Zoom The Zoom command the corresponding icon ql of the toolbar and the short cut F9 are used to zoom a section of the display range in the display The stack facility is also available here so the operator can toggle between the Undo Zooming and Redo Zooming commands The Zoom In function is active after the corresponding tool button is applied Consequently the mouse pointer converts automatically into the Zoom In pointer A inside the spectrum window The Zoom In function also becomes active when the mouse pointer is inside the Mass Spectrum Window and th
20. calibration buffer Apply Raw Changes the calibration constants to the Calibration ones that came with the raw data flexAnalysis 3 0 User Manual Version 3 0 2 35 Graphical User Interface GUI Bruker Daltonik GmbH 2 4 5 2 Internal If you want to adjust calibration parameter for a new calibration of a mass spectrum of a selected analysis use the Internal command or the corresponding icon A of the toolbar The Internal Mass Spectrum Calibration dialog Figure 45 opens During the calibration particular masses of the selected mass spectrum are assigned to corresponding reference masses of the loaded calibration list Calibration is only possible for MS or LIFT spectra that have been created with flexControl 3 0 Old LIFT spectra and FAST spectra cannot be recalibrated in flexAnalysis because the MS MS calibration is part of the acquisition system and thus part of the acquisition method in flexControl flexAnalysis offers three calibration strategies e Interactive Calibration automatic or manual using calibration lists MCLs e Smart that is an easy to use calibration procedure for optimum results The program reappraises the statistical frequency of occurrence of calibrants e Statistical calibration for peptide spectra The calibration strategy is saved in the flexAnalysis method and spectrum method but can also be changed A A Smart Mame Ref Mass Da Cur MassiDa Erri Err Da statistical Re
21. e flexAnalysis 3 0 and psClient 1 2 or psClient 1 3 The communication with ProteinScape 1 2 and ProteinScape 1 3 works The dialog of psClient is available After the command has been executed the dialog shown in Figure 90 appears ProteinScape x peClient 1 3 0 80 Server sgltestO Lagin Logout Send selected Spectrum gt Send Spectra from Spreadsheet Spectrum Achor Project Sample Gel Spot MSD ata Pssspectrum OOOO ne 1619 825 az 1046 545 1347 735 6000 4000 1252 644 2000 757 398 A B00 800 1000 1200 1400 1600 1800 miz a E Overlaid A Stacked A List Figure 90 Send to ProteinScape dialog Applying the Sent seestedSpectia gt button in the left upper corner opens the dialog of Figure 91 which is described in the Bruker ProteinScape User Manual flexAnalysis 3 0 User Manual Version 3 0 2 83 Graphical User Interface GUI Bruker Daltonik GmbH ps Export to ProteinScape on sqltest01 Standard Advanced Target as Gel Login J Filename D 10474 a3er13 0 921107060 test0_B711115Refipdatal111r Mass Spectrometer RNDTOPIS SpectrometerType Microflex Scout Code noo0000 0000000 0000000000 Position B7 Chip j Acquisition Date 006 07 117112 24 29 Processing Date dE Status of selected target mapping Cancel Figure 91 Example of an Export to ProteinScape dialog Send Spectra from 5 dsheet Se Applying the button opens a window to sele
22. level well above the baseline In these cases we can tell the software not to use the median as defined data point 50 but say use the value of data point 30 which is then supposed to be part of the baseline and not part of the data The algorithm ConvexHull3 constructs the baseline by fitting multiple parabolas to the spectrum starting with the large scale structures The baseline is then refined in an iterative way This is done in a way that the baseline is at least almost always below 2 62 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI the spectrum therefore the name Convex Hull baseline The baseline Flatness parameter influences baseline construction ConvexHull is usually preferred for a mass range of up to 10 kDa The advantage of this algorithm is that the baseline is a smooth function The algorithm TopHat constructs the baseline by means of morphology operators The baseline of the spectrum is obtained in two steps first each data point is replaced by the minimum value of the spectrum within n data points which gives the so called erosion Then within the same number of data points each value is replaced by the local maximum of the minimal values giving the opening of the spectrum which is the baseline The number of data points over which the minimum and maximum value is searched for is a function of the mass and is increasing in a way to get a good removal o
23. the pseudo slope exceeds a certain threshold a peak is regarded as peak The Sum algorithm makes internal use of the configurable baseline correction and noise calculation to select peaks and to calculate signal to noise ratios The configurable parameters are explained with the other algorithms me Edit Processing Method InternalCalibration ReadOnly EJES Mass List z 3 Find Mass List Find Edit E Processing Smoothing Baseline Sublr cti n Peak Detection Algorithrn Signal to Nowe Threshold Relative Intensity Threshold Minimum Intensity Threshold Maximal Number of Peaks Peak Width Baseline Subtraction Flatness MedianLeyvel Figure 67 Method parameters dialog box Mass List Find Sum flexAnalysis 3 0 User Manual Version 3 0 2 59 Graphical User Interface GUI Bruker Daltonik GmbH Mass List Edit E Edit Processing Method InternalCalibration ReadOnly El Mass List 3 Find Mass List Edit Edit a ll Use specific parameters for peak picking Smoothing Baseline Subtraction Checkbox not actrrabert Optimized settings for manual detection of weak peaks All Mass List Find parameters are used for annotation except the thresholds Thresholds are automatically set to zero Checkbox actrratect Define completly different parameters from Mass List Find for Mass List Edit Figure 68 Mass List Edit use Mass List Find as sensitive as possible The main differen
24. the selected analyses including the method Saves results of the selected analyses including the method to a user specific Saves results and method of all open analyses Signs an electronic record Shows the signature of a signed file Offers various possibilities to export data Displays print preview using the selected report layout Prints a report for the selected analyses Displays analysis and mass spectrum properties variable table Table 1 Features of the File Menu Feature File Menu Shortcut button commands gt Open Ctrl O Open single crri Shift O Analysis al Close Ctrl F4 Close All Load Unload Save Ctrl S Save As location Save All Shift Ctrl S Sign Electronic Record Show Signatures Export a Print Preview S Print Ctrl P Properties Alt Enter Exit Alt F4 Closes flexAnalysis 2 0 flexAnalysis 3 0 User Manual Version 3 0 Graphical User Interface GUI Bruker Daltonik GmbH 2 4 1 2 Open The Open command opens the Spectrum Browser dialog that allows selecting and loading spectra depending on several filter criteria it is not necessary to select the fid file to load a spectrum i Spectrum Browser pp TA Filter Spectra eegedateaemo O CO EE W MS iw LIFT LIFT converted _ Biotools_Flex FAST I FAST Segment D Unknown E e Biotools_Flex_BatchD ata E e warp2_discover mw E e Chip 0 i Spot G 015 Eo w Spot G O16 gt we Spot H 015 Spectr
25. tion Range fy Box Selection Figure 24 Contents of the Edit Menu Graphical User Interface GUI The Edit Menu contains commands to work with the appearance of spectra in the three main windows 2 4 2 1 Overview Table 2 Feature button i x Features of the Edit Menu Edit Menu commands Copy Paste Paste Special Delete Shortcut Ctrl C Ctrl V Del flexAnalysis 3 0 User Manual Version 3 0 Description Copies the selection context dependent and puts it on the Clipboard Inserts the contents of the clipboard Pastes methods method parameters or method scripts Deletes the selected mass spectrum 2 17 Graphical User Interface GUI Feature Edit Menu button commands Select All Select None View Analysis S E View Spectrum Set Color Default Color Set Selection Range Clear Selection E Box Selection 2 4 2 2 Copy Shortcut Ctrl A Ctrl D Ctrl Ins Ins Bruker Daltonik GmbH Description Selects all open analyses including all mass spectra Deselects all currently selected analyses and or mass spectra Hides Shows the selected analysis Hides Shows the selected spectrum except raw data Opens a palette of colors for manual allocation of a color to a spectrum Resets previous color assignments Selects a mass range in the Mass Spectrum Window Clears the current mass range selection in the active data window T
26. window You can save this sequence list together with the spectrum If you save and close a spectrum and re open it afterwards the same sequence list can be seen again 2 102 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI 2 4 12 Compass Menu Compass Help License Operator Lock All Applications CrriHtA lt K Compass Desktop Fil FlexContral Ctrl F11 Bio Tools Alt F12 GuditTrailviewer Figure 105 Content of the Compass menu 2 4 12 1 Overview Table 12 Features of the Compass Menu Feature Compass Menu Shortcut Description button commands License Enter the License number here Operator Changes the operator Locks all programs in case the Lock all Applications Ctrl Alt K Compass Security Pack is installed Compass Desktop F11 Opens the Compass Desktop FlexControl Ctrl F11 Opens flexControl BioTools Alt F12 Opens BioTools Opens the Audit Train Viewer in Audit Trail Viewer case the Compass Security Pack is installed 2 4 12 2 License The License command is used to open the Bruker Daltonics LicenseManager dialog box for viewing or adding removing licenses for Bruker products The License Manager looks the same in nearly all programs It is not necessary to enter a license for a flexAnalysis 3 0 User Manual Version 3 0 2 103 Graphical User Interface GUI Bruker Daltonik GmbH special Bruker program in the respective program it can be entered via the
27. 1 AA 1 f Compass _ _ for flexSeries a A Install Release Notes Contact Browse the CD Exit i DM PPU BRUKER DALTORIOS Figure 1 Compass CD flexAnalysis uses standard IBM PC and Microsoft Windows conventions to operate with windows menus dialog boxes and the mouse The operator is supposed to be familiar with the basic operation of the computer and with Microsoft Windows software Some general instructions are given here but if additional assistance is necessary regarding the basic use of a computer please review its accompanying documentation flexAnalysis 3 0 is able to operate in combination with the Bruker Compass Security Pack 1 2 This program can be installed prior to flexAnalysis or after it Main features of the Bruker Compass Security Pack are a user management electronic signatures and audit trailing This is different to the user management of the operating system The Bruker User Management is a tool that allows you to manage the access to Bruker applications and assign individual rights to users Depending on flexAnalysis 3 0 User Manual Version 3 0 1 1 Introduction Bruker Daltonik GmbH these rights the access of a user to features of Bruker applications is more or less restricted On saving data files the operator can electronically sign them if he has got this specific right Electronic signatures consist of the operator ID and his password Figure
28. 110 About dialog flexAnalysis 3 0 User Manual Version 3 0 2 107 Graphical User Interface GUI Bruker Daltonik GmbH 2 5 Analysis List Window The Analysis List Window or treeview is used to display one or more selected analyses and their related spectra 2 6 Mass Spectrum Window The Mass Spectrum Window or display is used to display a spectrum or spectra of one or more analyses prior selected in the Analysis List Window When a spectrum is displayed and the mouse pointer is within these window boundaries two interactive modes for convenient handling are available Mouse pointer the lt Ctrl gt key gt Edit mode section 2 4 3 3 Mouse pointer the lt Shift gt key gt Zoom mode section 2 4 9 7 flexAnalysis allows to toggle between both modes by alternately pressing the two keys the mouse must be in the Mass Spectrum Window 2 7 Mass List Window The Mass List Window or peaklist is used to display masses and peak parameters of a prior selected spectrum in the Mass Spectrum Window or the Analysis List Window Only one mass list is displayed at a time This list layout can be customized section 2 4 7 6 4 2 108 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI 2 8 Context Sensitive Menus 2 8 1 Getting Context Menus flexAnalysis offers three context menus accessible with the right mouse click Depending on the window of the GUI Treeview Display or Peaklist
29. 13 1363 829 1444 872 757 427 2465 308 stria a 1580 985 1612 980 pepmix0_4241211SRef 1619 920 1347 824 1635919 1651 908 1667 913 1764 963 1959 731 2049 177 2057 017 2076 125 1046 610 2093 196 2078 493 2093 204 2465 299 2465 308 a 3147 628 pepmid0_A2411115Ref 1347 745 1619 819 1046 551 2093 082 757 391 2465 176 3147 486 2000 2500 For Help press F1 pepmix 0_A24 3 Box Selection TOF User Figure 6 The Graphical User Interface The GUI is composed of the following items e Title Bar section 2 1 e Toolbar section 2 2 e Status Bar section 2 3 e Menu Bar section 2 4 e Analysis List Window section 2 5 e Mass Spectrum Window section 2 6 e Mass List Window section 2 7 flexAnalysis 3 0 User Manual Version 3 0 2 1 Graphical User Interface GUI Bruker Daltonik GmbH 2 1 Title Bar The Title Bar Figure 7 contains the name of the application and if one analysis is selected the title of the analysis file If a title is not set in flexAnalysis Properties dialog Figure 21 the first two comments shown in Figure 21 are displayed in the Title Bar The left end of the Title Bar contains the application s Control menu button The right end contains the Minimize Maximize and Close buttons just as all other Windows applications M flexAnalysis pepmix 20 06 2006 Ea lx Figure 7 Title Bar of f
30. 4 5 3 e Replace Calibration MS Spectrum refer to section 2 4 5 4 e Find Peaks refer to section 2 4 3 2 e Send Spectra to ProteinScape refer to section 2 4 10 6 2 34 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI Select one or more analyses in the treeview activate the check boxes of one or more processings and click the Start button to starts the processing A message appears reflecting the current state of the process The parameters that are used for processing can be set in the parameter editor for each analysis respectively Note Before using the Send Spectra to ProteinScape command you must have installed the access to your ProteinScape Server How to do this is described in the Bruker ProteinScape User Manual 2 4 5 Calibrate Menu Calibrate Annotation Method FAST AA Internal F Copy Calibration Replace Calibration Apply Raw Calibration Figure 44 Contents of the Calibrate Menu The Calibrate Menu Figure 44 is composed of various commands that have an effect on the calibration of MS spectra 2 4 5 1 Overview Table 5 Features of the Calibrate Menu Feature Calibration Menu Short Description button commands cut AM Internal Opens the Calibration dialog Copies the calibration of a selected analysis a OTAG into the calibration buffer of flexAnalysis Replaces the calibration of one or more Replace Calibration analyses with the content of the
31. 5H 46 H 34 O 11 926 4080 0 6130 14 00 odd Ic C20 HS2N 270 16 gl 926 4060 06le 6 30 even Ic 2elH5 N 200 21 1 926 4080 0 6244 3 00 odd a 19 H SE BT 2511 0 nil OSEA ANS A 2DAN z Ai 3s ale ATTO z Ey Done E My Computer Figure 94 Example of a report flexAnalysis 3 0 User Manual Version 3 0 2 93 Graphical User Interface GUI Bruker Daltonik GmbH 2 4 10 8 Macro Editor flexAnalysis offers two different ways for an automatic processing method scripts and macros Method scripts and macros are treated differently and designed in different editors The major difference is the range of application Method scripts are intended to operate on the single analysis they are assigned to while macros are independent of specific analyses The Macro Editor command is used to start the flexAnalysis Sax Basic Macro Editor Figure 95 fi Macro1 macro flexAnalysis design a ol x File Edit View Macro Debug Sheet Help Pea St Leelo LS o nu sl Mol gt m Object General Proc A 1 Sub Main A End Sub Figure 95 Sax Basic Macro Editor e This editor is an interactive design environment for developing testing and executing Sax Basic macros Sax Basic is Visual Basic for Applications compatible e flexAnalysis macros have a reference to Bruker flexAnalysis 3 0 Type Libraries which makes all flexAnalysis scripting objects available e Please refer to the editor help from the
32. 71928 236 246265571928 239 1275211836 cl 418027 443665011 1418027 443565011 1418133 73344116 c 0 047951555126750 0 0479515551261507 0 04596941 9042081 Type Tol Calibrants from PeptideCalibStandard mona Source file Date 2006 06 21 09 58 28 No of calibrants g Figure 47 Calibration Properties dialog Result Information Below the calibration list three result fields are shown the number of used calibrants the standard deviation before and standard deviation after the last calibration fit If no peaks are found during the automatic peak assignment the following message appears where possibilities are offered to correct the calibration flexAnalysis 3 0 User Manual Version 3 0 2 39 Graphical User Interface GUI Bruker Daltonik GmbH flexAnalysis Automatic calibrant assignment Failed You could try one of the Following Select a different Mass Control List Change the peak assignment tolerance Perform a new peak picking with different parameter settings Error Description Number of assigned Found calibrants too small Figure 48 Message to repeat automatic assignment with different conditions Smart Calibration Internal Mass Spectrum Calibration ES Calibration strategy Mass Control List smart Interactive E AS Mame Ref Mass Da Statist
33. 9912 C44 200 1 999 999907 C olH125N90 1 1000 000161 Co HislN2eoo 1000 000166 CYoH4 O6 1000 000239 i Hz6M50 43 393 999601 C17 H2 0N 12030 999 999596 C16H14 4N 190 35 q99 999590 CISHoW 460 26 999 999505 CHAN 250 16 1000 000415 C14H2N330 25 999 999580 3l HENS aza 1000 0004 0 Ci2Hn14 N11 0205 1000 0004 5 C 33H z0 N 4a a3 1000 000431 CB6QOHION10 16 999999410 CSFH4 hNeo il 999 999404 C4fH is N30 16 g99 999537 C465HiziN2loe 1000 000664 C4 6H151N100 11 399 999332 C Automatically locate monoisotopic peak Maximum number of formulas Check rings and double bonds Minimum Maximum Apply nitrogen rule Electron configuration both Minimam CH ratio 0 000 Masinum C H ratio 3 000 Show Pattern Figure 92 Generate Molecular Formula dialog Enter all the elements with their allowed minimum Min and maximum Max atom numbers on which the chemical formula calculation is based Formula calculation will start at the formula entered in Min and stop at the formula entered in Max Tips how to determine minimum and maximum formulas e Generally a CHNO distribution will be calculated If you want to exclude one of these atoms from the calculation you explicitly have to set it to 0 zero For example if there should be no N you enter NO e Sorting of elements is not needed 2 86 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI e Brackets can be used
34. E MEME MAME Ld Import Peak List Figure 74 Mass control list editor Peaks can be marked as Calibrants and or as Background peaks by activating the corresponding checkboxes Note e Only lists with at least one mass marked as calibrant appear in the drop down menu Mass control list e Only if peaks are marked as background peak they can be identified from a peaklist via the macro Identify Background Peaks section 2 4 3 7 e The Tolerance values are used for Background identification as well as for calibrant assignment e The columns Lock and Adduct are not yet in operation Apply to import an entire peaklist into the editor This list may be saved and also used as mass control list A pre installed i e write protected mass control list can be saved with another file name The new file is added to the MassControlLists directory and can now be used from all programs that work with MCLs The same mass control lists that are available in flexAnalysis are also available in flexControl Calibration tab and autoXecute Evaluation tab So if a list is changed in one of these programs this change is also available in the other programs For information about the other features of the Recalibration Masses tab please refer to section 2 4 5 flexAnalysis 3 0 User Manual Version 3 0 2 65 Graphical User Interface GUI Bruker Daltonik GmbH 2 4 7 6 4 Mass List Layout pepmix 0_A24 1 pepmix 0_A 2441 Method Para
35. Figure 40 Content of the Process Menu The Process Menu Figure 40 is composed of various commands to process spectra flexAnalysis 3 0 User Manual Version 3 0 2 31 Graphical User Interface GUI Bruker Daltonik GmbH 2 4 4 1 Overview Table 4 Features of the Process Menu Feature Process Menu Shortcut Description button commands idk Smooth Mass Smooth selected spectra Spectrum eo Sapir ai Subtract baseline from selected spectra Spectrum Baseline Undo All Processing Reverses all processing Batch Process Processes data in batch mode Process spectra Easy way of reprocessing spectra 2 4 4 2 Smooth Mass Spectrum The Smooth Mass Spectrum command and the corresponding icon Ls of the toolbar are used to smooth the selected mass spectrum The method parameter settings of the analysis are used for the smoothing operations section 2 4 7 6 1 2 4 4 3 Subtract Mass Spectrum Baseline The Subtract Mass Spectrum Baseline command and the corresponding icon be of the Toolbar are used to perform a baseline subtraction for the selected spectrum or spectra The method parameter settings of the analysis are used for the baseline subtraction operation section 2 4 7 6 1 2 4 4 4 Undo All Processing The Undo All Processing command is used to return to the starting point before any processing took place The raw data file fid file is loaded and displayed in the Mass Spectrum Window Note that Undo All Processing does not ca
36. GUI Fille Edit Mass List Process Calibrate Annotation Method FAST Sell Sale Bax A aw autorun more protein aso 61641 loaded Y ly 1SRef O ilh LA iha LA ihan r S 4 autorun_werify40_G8 1 isis illh 15Ref Figure 14 Loaded and unloaded spectra in the treeview If you want to open a large number of spectra in the flexAnalysis treeview without loading them all at once you may open them with the Spectrum Browser Open command Figure 12 In this dialog you will find the checkbox Load all selected spectra at the bottom For loading a lot of spectra the checkbox should not be activated Otherwise if you only want to load few spectra at once activate this box 2 4 1 7 Save The Save command the corresponding icon of the Toolbar and the short cut Ctrl S store the current processing state of the selected analysis including the processing method The Save command can be applied to several selected analyses at a time after these have been marked in the treeview with the lt Shift gt or lt Ctrl gt keys of the keyboard thus following Windows conventions 2 4 1 8 Save As The Save As command is used to save the selected analyses including the method with a new name and to a new location 2 4 1 9 Save All The Save All command or the short cut Shift Ctrl S stores the result spectra of all opened analyses at once including the processing methods flexAnalysis 3 0 User Manual Version 3 0 2 9
37. License Manager of other Bruker programs Bruker Daltonics LicenseManager New license key Existing licenses License key Walid Product name unlimited BioTools 3 1 unlimited BioToole 3 1 RapiDeNowa unlimited Compass Security Pack 1 0 unlimited Compass Security Pack 1 2 unlimited flexdnralysis 3 0 unlimited fle Cortrol 3 0 24367 76BCO3MLPLL 220606 06 22 06 Flezlmaging 2 0 2 01 834FCOBBDSLA 130706 07 13 06 GenoT ools 2 0 OligoQl 2fO1 8230 C05E3U006 130706 077 13 06 GenoTools 2 0 SNP unlimited MolecularFormulaGenerator 1 0 Figure 106 The License Manager 2 4 12 3 Operator The Operator command is used to log on as new operator The new operator name appears in the Status bar at the bottom of the GUI In case the Bruker Daltonics UserManagement is installed additionally a password is requested If a user logs on with the Operator command the user automatically changes for all other Bruker programs that are currently open and run with the UserManagement In flexControl this can be seen because the Select Method dialog comes up 2 4 12 4 Lock all applications The Lock all Applications command and the short cut Ctrl Alt K lock all the Bruker applications that are currently open depending on the lock time that is defined in the UserManagement Additionally the Unlock dialog opens If one program is locked manually or via timeout all programs that run with the UserManagement are locked since they all u
38. User Interface GUI number of nitrogen molecules must be odd too Things are just the other way if you have radical ions Instead of the common definition which considers the production of radical ions by El techniques were are considering the production of pseudo molecular ions M H M Na M H resulting from soft ionization methods To be more precisely the number of nitrogen atoms is calculated to be even if the nominal mass plus the number of deuterium atoms plus the charge state is even Applying this rule will result in only formulas complying with the nitrogen rule being generated Otherwise also formulas not complying with this rule may be generated Default is unchecked Electron configuration Select the kind of electron configuration to be applied e even Generates only molecular formulas having an even electron configuration e odd Generates only molecular formulas having an odd electron configuration e both Default Generates molecular formulas of both electron configurations The electron configuration is calculated under the assumption that we have molecular ions The electron configuration is even if n atom x valency atom z ion is even Minimum C H ratio Enter the minimum C H ratio to be applied Default in 0 Maximum C H ratio Enter the maximum C H ratio to be applied Default in 3 Generate Generates for the mass range that is specified by the Measured m z and Toler
39. ally the respective mass range corresponding to the selected reference mass It is possible to choose between some relative and absolute Zoom ranges Note You can use the mouse wheel to select the next reference mass or to go back to a previous one This simplifies the manual assignment Peak Assignment Tolerance The tolerance defines a mass interval that is used for the calibrant assignment If the difference between a reference mass and a peak in the peaklist is smaller than the tolerance the peak is assigned to the calibrant If more than one peak of the peak list fits this criterion the peak with the smallest mass difference to the calibrant is assigned to the calibrant Calibration Fit Order Selecting this option performs a quadratic or linear calibration calculating the coefficients Co Cy and C2 For linear calibrations c2 is set to zero N 38 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI The Properties dialog shows the current state of the calibration and some general calibration information If spectra have been acquired with a flexControl HPC method the check box High Precision Calibration applied at the bottom is activated read only check box The decision whether the HPC calibration is used or not is done via the Options dialog section 2 4 10 10 4 from the Tools menu Calibration Properties Fit Result Current lado saved Preview gt of P35 2462655
40. an be copied into the clipboard Afterwards they can be pasted to another analysis 1 Select an analysis in the Analysis List Window 2 Select Copy Calibration The command copies the calibration of the selected analysis into the calibration memory of flexAnalysis Afterwards it can be pasted to other analysis analyses Only MS finger print calibrations can be copied and transferred into the calibration buffer 2 42 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI 2 4 5 4 Replace Calibration The Replace Calibration command transfers the calibration from the calibration memory to the selected analyses Precondition A TOF calibration i e the constants Co Cy and c2 of an analysis have already been copied into the calibration memory of flexAnalysis 1 Select on ore more analysis analyses in the Analysis List Window 2 SelectReplace Calibration The calibration constants are transferred Before a calibration is transferred from one analysis to another one it is checked whether the high voltages of the ion sources 1 and 2 the lens and reflector voltages of the two analyses are identical within a range of 0 01 V If this is not the case a warning message appears Press No if you don t want to transfer the calibration and Yes if you want to apply the external calibration despite of the mismatch of the calibration voltages This describes the manual way to do a calibration an
41. ance values molecular formulas that meet the current parameter settings and enters them along with corresponding data in the formula list If Show Pattern is already turned on the pattern of the first formula in this list will be displayed below the original spectrum and entered in the Analysis List as a subentry of the original spectrum List of generated molecular formulas Lists the molecular formulas that have been calculated for the specified Measured m z value according to the current parameter settings flexAnalysis 3 0 User Manual Version 3 0 2 89 Graphical User Interface GUI Bruker Daltonik GmbH For each formula the following set of data is calculated that includes for instance various error values rankings and statistical values Consecutive number of the formula MF Molecular formula m z m z of the formula err ppm Deviation between the measured mass and the theoretical mass of the selected peak dbl eq Number of rings and double bonds in the formula refer to page 2 88 N rule Indicates whether the nitrogen rule is fulfilled e Indicates whether the electron configuration is even or odd The electron configuration is calculated under the assumption that we have molecular ions The electron configuration is even if n atom x valency atom z ion is even Various rankings The ranking is a number between 1 and the number of formulas 1 stands for the best formula candidate according to th
42. and is used to clear a selected range in the Mass Spectrum Window 2 4 2 14 Box Selection This function determines the selection mode If the box selection is switched on both the mass range and the intensity range are selected If the box is switched off only the mass range is selected 2 4 3 Mass List Menu Mass List Process Calibrate Annotation f AL Eind F5 fh Edit Ctrl Th Edit Direct ctrl alt Edit Mass Control List Distance in Mass Listis Figure 30 Contents of the Mass List Menu The Mass List Menu Figure 30 is composed of commands to work with mass lists 2 22 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI 2 4 3 1 Overview Table 3 Features of the Mass List Menu Feature Mass List Shortcut Description button Menu commands A Find FS Automatically creates a mass list for the selected spectrum spectra De Activates the Edit Mass List mode to edit MS ect the mass list of the selected spectrum Th Edit Direct Ctrl Alt Adds a m z intensity pair to the peaklist x Shift Ae Clear FS Clears the mass list of the selected spectrum Opens a list with masses that have been MS MS List selected from the MS peaklist for manual or automatic LIFT measurement in flexControl Edit Mass Control List Opens the Mass Control List Editor o d Identifies background peaks on the basis of a 9 Mass Control List that has to be selected here Peaks moe Remo
43. ands Set Display Sets the data display range for the active Range data window al Zoom F9 Zooms the selected data range IQ Zoom X Shift F9 Zooms only in x direction Undo al Zooming Alt Left Undoes the last zooming step Ey pede Alt Right Redoes the previous unzoom step Zooming l Toolbars Shows hides or customizes toolbars e Status Bar Shows or hides the Status Bar 2 4 9 2 Auto Scaling The Auto Scaling command the corresponding tool button and the short cut Alt F9 are used to toggle abscissa and ordinate in the Mass Spectrum Window between auto scaling and manual scaling On selecting this command the auto scaling mode takes effect or not Auto Scaling means that the display range of both axes is automatically adjusted to display the maximum measured values When this mode is active the corresponding tool button in the Toolbar is highlighted Note When using this command in the Mass Spectrum Window the operator cannot individually set the auto scaling status for the ordinate or abscissa Any changes of the abscissa and or ordinate display range will deactivate auto scaling of the corresponding axis axes Auto scaling can be activated and de activated with double click in the display or beneath and next to the x and y axis Additionally the button and can be used 2 4 9 3 Scale to Entire Spectrum The Scale to Entire Spectrum command and the short cut Ctrl F9 are used to return from a zoomed section of
44. annnonannnnnannnonnnnnnancnnnnnninas 2 79 ZAAOD BIO TOUS ol eii Latas 2 81 ZAAO PON TOS ii a li ia a eA 2 82 24 10 35 PLANA CA e ol e E 2 82 2 4 10 6 Generate Molecular fOrMulas c coccccconcccccncncocnncconnnonnnnnonnnnnnnnnononononos 2 84 ZARA Macro E e LaS 2 94 2410 39 CUSIONMUZS da 2 95 ZNO FOIOS A tacto 2 96 ZAIDA IS a ait teen 2 97 ZA NO ADS e a do 2 97 LANDS EDO a tide tote do 2 98 ZANO O Calla att 2 99 Zu E RO ON 9 0 S cess ser OPI E caret ne ti acen ances anos cae te E EE E eee 2 99 24 10 11 ASIVGCODC PUGS Sisi 2 100 2 4 10 12 Send Spectra to ProteinSCAape ccccoocccccoccccccccncccnnnnconnnnonnnononononononononos 2 100 ZA NVIROOWAMICN Mis AA A 2 101 ZA QVEWICW a A atari 2 101 24 112 Analysis LSE sails dare ee ies cea aed ee ans a eee 2 101 241 13 MASS ECU o 2 101 24114 MassLiStaiann Seas wien ened Ree 2 102 DONS Sequence stos a ieee wane eee ee 2 102 24 12 Compass Menu sc2ui tee did 2 102 ZAA2 OVV OW cc das 2 103 ZANZ 2 LICEON S Oinen aden eRe teehee ace neta ENE eae oes 2 103 ZAANZD Operator areas assets aaa 2 104 24124 LOCK all aDPIICANONS ico 2 104 ZAAZS Compass DESK aia 2 105 2A I2 FICXGONMMOL ariora a a eta 2 105 ZZ A A iewantenede 2 105 ZG N220 AMO Tal WICWON a 2 106 ZAS Heli Met io sidad 2 106 DANO COVE Wa a a a R 2 107 2A o2 Hep ODIOS ner iria 2 107 2 4 13 3 About Bruker Daltonics flexAnalySiS ccooooncoccconccococococononcnnconanonoos 2 107 flexAnalysis 3 0 Us
45. anual Version 3 0 Graphical User Interface GUI Bruker Daltonik GmbH The Mass Spectrum tab Figure 22 is not editable It displays groups of acquisition and processing parameters of the current analysis The group arrangement and the distribution of the parameters have been done according to flexControl The flexAnalysis parameter groups for example have the same names as the tabs in flexControl where the acquisition parameter can be adjusted pepmid0_A2413 Analysis Properties Type Tof Calibrator Mass Control List Source file Date 2006 06 20 10 36 01 Mo of Calibrarits cl 239 128 cl 418135 733 Ce 0 046 20000 40000 60000 SOOO Time ps Figure 23 Contents of the Calibration tab The Calibration tab Figure 23 can be used to verify the calibration constants Additionally some general information about the calibration are shown the possibly used Mass Control List and in case the calibration constants have been copied from another spectrum the sample name of this spectrum 2 4 1 16 Exit The Exit command and the short cut Alt F4 are used to close flexAnalysis However before closing a dialog may open and ask you to save modified analyses 2 16 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH 2 4 2 Edit Menu Edit Mass List Process Calibrate An Copy Ctro Select All Chri A Select Mone Chrl D View Analysis Ctrl Ins View Spectrum Ins Set Color d Default Color Set Selec
46. arked with a red arrow indicating that the calibrant is in use but not assigned If you have chosen a correct setting for your zoom range your calibrant peak is displayed in the Mass Spectrum Window 5 Select your calibrant peak in the Mass Spectrum Window with a mouse click on the top of the peak If the assignment was successful the current mass entry and the error entry are filled with the calculated values The row is now marked with a green arrow that indicates a successful assignment The calibration constants displayed in the dialog Co C2 are also immediately updated 6 Whether the calculation based on the new assignment improved the calibration can be evaluated by checking the standard deviation values before and after calibration The calibration constants itself can be checked by pressing the Check Calibration button 7 Repeat steps 4 and 5 by using the mouse wheel until you have either assigned all the entries in your reference mass list or all the calibrant peaks in your spectrum When the user wants to remove a calibration point from the table he simply clicks with the left mouse button onto the corresponding line Be aware that the button Clear removes all the previously matched masses The user can also verify whether a spectrum is calibrated correctly within the properties dialog A II flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Appendix A 2 High Precision Polynomal Calibration HPC
47. ated from the peak finder It is useful for example for Imaging runs where the fuzzy control is de activated and spectra that only consist of electronic noise are acquired They show a flat baseline and some spikes with small intensities that will be annotated and will distort the picture The Minimum Intensity Threshold allows ignoring these Maximal Number of Peaks This is the maximal number of peaks that shall be detected by any of the three detection algorithms In case of SNAP this value is an additional filter regarding the Quality factor for all other algorithms the peaks with the highest intensity will be selected Additionally each peak detection algorithm has also some special parameters that can only be adjusted if the respective algorithm has been chosen from the drop down list Peak Detection Algorithm Centroid The peak finding mechanism for Centroid Figure 63 uses the first and second derivative to detect a peak For the peak position a specific cut off level above the baseline Percent Height has to be specified A value around 80 is recommended The baseline is subtracted automatically when using Centroid The baseline subtraction algorithm has to be selected on the Mass List Find tab Note The algorithm chosen here is not used for the real baseline subtraction Other special Centroid parameters are Peak Width The parameter Peak Width is no threshold but an expectancy value Peaks having this width are expect
48. ator jadmin Program Action ES Object lon PC 10 493 29 155 Old State New State Ea Reason Figure 108 DateTime Audit Trail Viewer 2 4 13 Help Menu Help de Help Topics Fi T About Bruker Daltonics FlexAnalysis Figure 109 Contents of the Help Menu 2 106 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI 2 4 13 1 Overview Table 13 Features of the Help Menu Feature Help Menu commands Shortcut Description button ae Help Topics F1 Opens the help file p About Bruker Daltonics flexAnalysis 3 0 Opens the About Window 2 4 13 2 Help Topics The Help Topics command the corresponding icon e of the Toolbar and the short cut F1 open the flexAnalysis online help to get information about specific topics 2 4 13 3 About Bruker Daltonics flexAnalysis This command opens the About dialog Figure 110 that displays the version number of your flexAnalysis version with the copyright notice the user specific license information and contact information of Bruker Daltonik GmbH About Bruker Daltonics flexAnalysis Bruker Daltonics flexAnalysis 3 0 flex4nalysis Version 3 0 Build 45 Copyright 2001 2004 Bruker Daltonik GmbH This product is licensed to User BOALDE License key Sub Products ACO 301020 NTE 148411 Phone 49 421 205 345 Fax 49 421 2205 5390 E Mail maldi w supporttibdal de WTA http A bal de Look Figure
49. atures that are available in these menus can be found also as buttons in the button bar or as entries in the context menus of the three main flexAnalysis windows File Edit Mass List Process Calibrate Annotation Method FAST View Tools Window Compass Help Figure 10 Content of the Menu Bar flexAnalysis 3 0 User Manual Version 3 0 2 3 Graphical User Interface GUI Bruker Daltonik GmbH 2 4 1 File Menu File Edit Mass List Process Calibrate Annotation Method FAST View Tools win Gar Open chro Open Single Analysis Cbrl ShiFk 0 B Close Ctrl F4 Close All Unload Ei gave Ctrl 5 Save S Bl Save All Ctrl Shift 5 Export d A Print Preview 5 Print Ctrl F Properties Alt Enter 1 DD TA DemolBiotools Plex BatchDatawarpe veriPyl1 6711115RefF 2 DADA TA DemolBiotools Plex BatchDatalwarpz_veriPy10_ H811115Ref 3D004T74M DemolBiotools Plex BatchDatawarpz_veriPy10_H711115Ref Exit Alt F4 Figure 11 Contents of the File Menu The File Menu is composed of commands to open close and save data handle the user management and adjust the printout 2 4 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI 2 4 1 1 Overview Description Opens the Open multiple spectra dialog Opens one selected analysis Closes the selected analysis or analyses Closes all analyses Loads an analysis in the treeview Unloads an analysis from the treeview Saves results of
50. ble for high mass precision instruments Switching between 1 and 1 makes a difference of two electrons or 1 1 mDa which means 2 ppm for a molecule with 548 Da Automatically locate monoisotopic peak Select this option if the monoisotopic peak corresponding to the Measured m z should be located automatically The algorithm will search to the left and right of the selected Measured m z for appropriate sum formulas with their corresponding monoisotopic peaks To use this option select the largest peak of the pattern If the search was successful the found formula s for the corresponding monoisotopic peak s is entered into the results list In formulas containing a monoisotopic peak which is different from the selected peak the monoisotopic mass is slightly different by a few Da from the selected mass flexAnalysis 3 0 User Manual Version 3 0 2 87 Graphical User Interface GUI Bruker Daltonik GmbH The mass error nevertheless is calculated for the selected peak and the exact theoretical mass of that peak based on the formula from Alan L Rockwood et al J Am Soc Mass Spectrom 2004 15 12 21 Default is cleared Maximum number of formulas Enter the maximum number of molecular formulas to be calculated at the most The formulas with the smallest relative error based on the measured m z will be picked out Default is 200 Check rings plus double bonds Select this option if possible molecular formulas should be check
51. cal User Interface GUI 2 4 9 10 Toolbars The Toolbars command opens the Toolbars dialog box Figure 86 This feature is used to show hide or customize Toolbars This is simply done by un checking the corresponding checkbox At the same time the related part of the Toolbar appears or disappears on the GUI Toolbars Toolbars El h denu bar A Customize Rese ae Help Show Tooltips Cool Look Large Buttons Figure 86 The Toolbars dialog box The list box Toolbars shows all the Toolbars currently defined defaults as well as custom created toolbars Any toolbar has a check box in front of it indicating its current display status The button New allows to create a user defined Toolbar Hew Toolbar Toolbar name Figure 87 New toolbar dialog flexAnalysis 3 0 User Manual Version 3 0 2 17 Graphical User Interface GUI 2 4 9 11 Status Bar Bruker Daltonik GmbH The Status Bar command is used to show or hide the Status Bar at the bottom of the GUI When the Status Bar is visible it is indicated by a highlighted check mark in front of this command 2 4 10 Tools Menu Tools Window Compass Help El ReportDesigner Se ProteinScape Generate Molecular Formulas CS Macro Editor Customize 3 Options Colors Glycopeptides j Send Spectra to Protein cape Figure 88 Contents of the Tools Menu 2 78 flexAnalysis 3 0 User Manual Versio
52. ce between the Mass List Edit tab and the Mass List Find tab is the checkbox Use specific parameters for peak picking If this checkbox is not selected Figure 68 all parameter settings that are currently selected in Mass List Find are used for the Mass List Edit procedure except the Thresho d parameters which are set to zero This has the effect that the peak finder which was used for a Mass List Find is as sensitive as possible in order to detect additional peak with Mass List Edit manually t Edit Processing Method InternalCalibration ReadOnly E Mass List a r Find Mass List Edit Edit a ll W Use specific parameters for peak picking Smooth 7 Ai Peak Detection Algorithm Sur e Baseline Subtraction Signal to Noize Threshold fi Relative Intensity Threshold o 4 Minimum Intensity Threshold 0 Maximal Number of Peaks Peak Width 0 1 mz Baseline Subtraction edian a Flatness 08 MedianLevel 0 5 Figure 69 Mass List Edit define special values 2 60 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI If the checkbox is activated Figure 69 different parameters can be selected The Edit field Maximal Number of Peaks is deactivated because only one peak is detected at a time Processing Smoothing The settings for Smoothing process section 2 4 4 2 can be adjusted in the secti
53. cess for LIFT spectra Certainly this can be changed especially if the user does not want a script running for every spectrum that is newly loaded 2 4 8 FAST Menu FAST View Tools Window Compass Paste Segment Spectra Change Parameters Figure 79 Contents of the FAST Menu The FAST Menu offers FAST specific operations and is only available if a FAST segment analysis or FAST analysis is unambiguously selected For all other spectra types the menu entries are not selectable To display FAST segments with their correct mass values flexAnalysis is capable to recognize the analysis type during loading 2 4 8 1 Overview Table 8 Features of the FAST Menu Feature Mass List Menu Shortcut Description button commands Paste Segment Pastes all segments to one spectrum Spectra Change To change parent and segment Parameters parameters Delete Segment Deletes segment spectra from hard disk Spectra flexAnalysis 3 0 User Manual Version 3 0 2 69 Graphical User Interface GUI Bruker Daltonik GmbH 2 4 8 2 Paste Segment Spectra The Paste Segment Spectra command is used to paste FAST segment spectra to one linearized FAST spectrum as Raw data fids are used in the paste process The following rule is applied for pasting First flexAnalysis uses the penultimate segment n 1 until mass Mmax and then follows the last segment n without overlapping both segments Intens Figure 80 Exampl
54. commands E Analysis List Alt 1 Fades in out the Analysis List Window m Mass Spectrum Alt 2 Fades in out the Mass Spectrum Window Mass List Alt 3 Fades in out the Mass List Window Sequence List Fades in out the Sequence List Window 2 4 11 2 Analysis List The Analysis List command the corresponding icon of the toolbar E and the short cut A1t 1 display or remove the analysis list from the GUI On displaying the list a highlighted check mark is shown in front of this command 2 4 11 3 Mass Spectrum The Mass Spectrum command the corresponding icon of the toolbar ha or the short cut Alt 2 toggle the Mass Spectrum Window and the Bruker symbol On displaying the spectrum a highlighted check mark is shown in front of this command flexAnalysis 3 0 User Manual Version 3 0 2 101 Graphical User Interface GUI Bruker Daltonik GmbH 2 4 11 4 Mass List The Mass List command the corresponding icon of the toolbar a and the short cut Alt 3 display or hide the mass list from the GUI When the Mass List Window is shown the tool button shown in front of this command is highlighted 2 4 11 5 Sequence List The Sequence List command the corresponding icon of the Toolbar E and the shortcut Alt 4 fill this wndow with sequence tags which you can create with the annotation feature section 2 4 6 If there is a gap in the sequence tag a new entry with the next abbreviation of the currently chosen annotation will occur in this
55. ct a spreadsheet sequence to export the related data files to the ProteinScape server 2 4 10 7 Generate Molecular formulas The Generate Molecular Formula command is used to calculate all kinds of molecular formulas of a measured m z value taken from a highly resolved mass spectrum You can preserve a pattern s of a calculated formula s by saving it them with the original spectrum as an xml file The basic result elements of a Generate Formula operation may be displayed in HTML format and printed out The calculation of elemental compositions of molecular and fragment ions need exact mass determinations from a highly resolved mass spectrum The aim is to determine all theoretically possible elemental compositions of a mass within a specified error tolerance 2 84 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI The higher the measured mass the more elemental compositions are possible For example up to a mass of 1000 Da approximately 66 000 compositions are theoretically possible solely consisting of C H N and O For masses up to 2000 Da this value increases up to 520 000 Additional elements that may be found in the compositions will extremely increase the number of compositions Because of this large number of possible combinations it is very time consuming to trace all of them and compare the resulting mass with the measured mass Therefore the number of solutions needs to be r
56. ct calculation of the monoisotopic masses Monoisotopic masses are the basis for database search and DeNovoSequencing which allows elucidating structure and sequence of a particular peptide if database search for MS MS data failed SNAP calculates the isotopic distribution of a given mass charge and mean molecular constitution and uses the resulting isotopic pattern for a nonlinear fit that delivers the monoisotopic mass in the end 2 56 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI E Edit Processing Method InternalCalibration ReadOnly Mass List E Mass List Find Edit Processing Smoothing Rasele Subtraction Peak Detection Algorithm Signal to Mowe Threshold wn a i ot Relative Intensity Threshold Minimum Intensity Threshold Maximal Number of Peaks Quality Factor Threshold 000 un a o ale SMAP average composition Averagine E a a Fragment Peak width SILE Mass Difference s H I SILE Partner SH Threshold Baseline Subtraction Median Flatness de MedianLevel Figure 64 Method parameters dialog box Mass List Find Snap Quality Factor Threshold The parameter Quality Factor Threshold describes the reliability of a peak lt is a composition of peak intensity and consistency with known isotopic patterns It is not possible to give a recommendation for this value the best is to have a look on it in some represe
57. d that is available in the Mass List context menu section 2 8 Masses from an MS peaklist that have been added to the MS MS List can be sent to flexControl for manual L_sendtoflexcontrel jy or automatic _Append to autoxecute Run J LIFT measurement certainly also Run method on current can be used in flexControl flexAnalysis 3 0 User Manual Version 3 0 2 25 Graphical User Interface GUI Bruker Daltonik GmbH To add masses from the peaklist to the MS MS list right click on the desired mass and choose Add Mass to MS MS List This can be done for several peaks from the same spectrum and for several spectra from the same target After masses have been added to the list it can be opened with the MS MS List command that is shown in Figure 32 Peak masses and spot numbers are listed The whole list can be cleared with the button single masses can be removed with the L_2eete _ Jbutton The button is used to send the whole list to flexControl lt is not necessary to have flexControl opened before In flexControl LIFT tab the masses of this list are then shown as selection list so there is no need to type in the masses manually one by one MS MS List Peak Mass Spot Mo 1479 853461 H7 D 1567 78616 H7 D 027 48087 Hi 1667 04369 Send to flexContral Figure 32 MS MS List with masses selected for LIFT measurement With the button the chosen masses can be added to the AutoXecute
58. d transfer it to a set of sample spectra Automatically can this be done within an AutoXecute run with two pre installed methods CalibratePeptide Standards FAMSMethod and ExternalCalibration FAMSMethod The first one is applied to spectra acquired on a calibration sample the script of this method cares for the calibration process and the copying of the new calibration constants The second method is applied to spectra that are acquired on the sample spot after the calibration measurement The script cares for the transfer of the newly calculated calibration constants 2 4 5 5 Apply Raw Calibration The Apply Raw Calibration command is used to reset the calibration to those values which were originally stored during the acquisition The peak list will be re calculated This command is only applicable for MS and FAST segment spectra After applying and before executing this command a warning message Figure 51 appears flexAnalysis A The raw calibration might possibly differ significantly From your current calibration Are you sure that you want the raw calibration applied Figure 51 Warning before applying the raw calibration flexAnalysis 3 0 User Manual Version 3 0 2 43 Graphical User Interface GUI Bruker Daltonik GmbH 2 4 6 Annotation Menu Annotation Method FAST wiew Tc t Annotate Alt ri Clear t Show Mass Difference fit Preview Assignments 2 Edit Building Blocks Figure 52 Content of the A
59. dian and TopHat me Edit Processing Method pepmix 0_A24 1 a Mass List y 3 Find Baseline Subtraction Edit E Processing Smoothing Baseline Subtraction Select Algorithm Median Flatness MedianLevel Figure 71 Method parameters Baseline Subtraction The algorithm Median is determined through the parameters Flatness and MedianLevel A spectrum that will be baseline subtracted is divided into a number of segments The minimal number of segments is 1 i e Flatness equals zero The maximal number of segments is 100 which corresponds to a Flatness of 1 A strong baseline drift is associated with high Flatness values The parameter Flatness influences the number of parabolas used to explain the baseline and the flatness of the resulting spectrum which is obtained by subtracting the baseline from the spectrum The larger the flatness value the finer the baseline will approach the spectrum The following values are recommended for Flatness 0 0 for totally flat spectra without baseline drift 0 1 for protein spectra and 0 8 for digest spectra recorded in reflector mode The Median Level allows shifting the baseline relative to the data By definition if we have 100 data points the median a common statistical size is the value of data point 50 after the data points are sorted by their y values If we have a very dense spectrum then the sorted data point 50 can already be something that is in the data
60. e lt Shift gt key is pressed flexAnalysis 3 0 User Manual Version 3 0 2 75 Graphical User Interface GUI Bruker Daltonik GmbH 2 4 9 7 Zoom X The Zoom X command the corresponding icon Al of the Toolbar and the short cut Shift F9 are used to zoom spectra or peaks in x direction The ordinate value remains unchanged 2 4 9 8 Undo Zooming The Undo Zooming command the corresponding icon al of the Toolbar the short cut Alt Left Arrow and a simple mouse click into the Mass Spectrum window when the Zoom mode is active are used to reverse the last zooming step that was performed flexAnalysis is able to put the last twenty zooming steps of the current session into a particular stack So the operator can toggle back to previous zoom steps by continuously selecting Undo Zooming until to the desired display When the last step is taken from the stack flexAnalysis deactivates this function 2 4 9 9 Redo Zooming The Redo Zooming command the corresponding icon EY of the Toolbar or the short cut Alt Right Arrow are the counterpart of the Undo Zooming function and are used to retrieve a previous step performed with the Undo Zooming command On selecting Redo Zooming the display range toggles to the zooming step that was performed previously Also here flexAnalysis stacks up to twenty last zooming steps performed during the current session 2 76 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphi
61. e Menu The Report Layout files are installed in a folder named Report Layouts which is located in the methods directory normally D Methods 2 4 10 4 BioTools BioTools is a Bruker program for peak annotation of aminoacids whose spectra are already processed e g with flexAnalysis BioTools uses these spectra and peaklists for DeNovoSequencing on internet searches The BioTools command is active if BioTools is installed in a compatible version on the same computer and if an analysis is loaded and marked in flexAnalysis The command is used to save the currently active spectrum open the BioTools program and transfer the spectrum to BioTools flexAnalysis 3 0 User Manual Version 3 0 2 81 Graphical User Interface GUI Bruker Daltonik GmbH 2 4 10 5 PolyTools PolyTools is a program from Bruker for determination of monomers end groups and cations Spectra must be already processed e g with flexAnalysis since PolyTools needs these spectra and peaklists If isotopes are resolved in the measured spectrum lines belonging to the isotope distribution are identified and can be reduced to amino isotopic series For every isotope series or the reduced monoisotopic series the average molecular weight polydispersity and degree of polymerisation are determined It is also possible to enter repeat unit end groups and cation manually either in addition to or replacing the values found automatically New stick spectra calculated from al
62. e individual value rank dintens Ranking according to dev Intens rank dintensVarNorm Ranking according to dev IntensVarNorm rank dMeanMass Ranking according to dev MeanMass rank dMass Diffs Ranking according to dev dMass Diffs rank dCombined Ranking according to dev Combined rank dMeanMass Error Ranking according to dev dMeanMass Error rank dm z Ranking according to dev m z Various statistical values dev Intens Root mean square RMS of the normalized intensity of the peaks The peaks are normalized so that the sum of intensities is one An offset which is the same for all peaks is added to reduce an intensity offset which might remain after optimizing the digitizer setting dev IntensVarNorm Same value as dev Intens but the peaks are weighted by the square root of the intensity to take into account the ion statistic dev MeanMeas RMS of intensity weighted mass differences between the measured masses and the theoretical masses for the given formula dev MassDiffs Intensity weighted RMS of the difference between the measured and theoretical peak distances between peaks being 1 or 2 Da apart Not only 2 90 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI the monoisotopic mass and the relative intensities of the peaks depend on the formula but also the distances between the various peaks With instruments like the microTOF and especially the FTMS instruments this differe
63. e of a segment spectrum before and after pasting The paste procedure results in a new analysis that is also shown in the tree view This analysis contains all spectra which arise from multiple use of the Paste command referring on the same FAST segment analysis 2 70 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI Analysis List SM ACTH_10_57_PSD_2465 20_0 H9 2 2 10 O lit 15Het 2 O lit 5Hel O ll 35Ref Raw 2 q O h h 45hel o O h h ooFet 2 O lit b5Hel o O lit 5hel 2 e O lit os het O ll 95 Ref Raw ve i TOSRef Raw i ve LJ 115Ref Raw ve Cll 125Ref Raw va Cll 135Ref Raw e py 145 Ref Raw A lay 27H_10_57_PS0_2465 20_0_H3 H bel lh fast Raw a Wl Fast Raw Figure 81 New analysis containing two pasted spectra Results are not automatically saved after manual pasting On saving an analysis which contains the result of a paste procedure a result spectrum of this analysis is saved in a directory samplename lt parentmass gt FAST FAST Only the pasted spectrum shown in bold letters in the tree view will be saved with the Save command Note If there is already a spectrum in samplename lt parentmass gt FAST FAST it will be overwritten because on the hard drive at any time only one result spectrum is allowed 2 4 8 3 Change Parameters The Change Parameters command is used to change the parent mass the FAST segment parameters per segment for example the seg
64. e parameter Offset specifies the distance among the shown spectra These parameters determine how spectra appear in the Stacked View of the display The parameter Windows of the group box List determines the number of windows displayed in the List View 2 4 10 10 2 Labels Options Lalik Display Labels Export Calibration Density Non ovelapping O Non overlapping with tics Overlapping O Tics only Orientation Horizontal O Vertical Figure 100 Option to specify the appearance of the peak annotation flexAnalysis 3 0 User Manual Version 3 0 2 97 Graphical User Interface GUI Bruker Daltonik GmbH The Label tab Figure 100 contains two groups of radio buttons to specify how peak labeling shall be performed Density e Non overlapping prevents overlapping of the labels with the spectrum curve Labels are not shown if they would overlap with the spectrum or neighbouring labels e Non overlapping with tics prevents overlapping of the labels and shows tics on the top of a labeled peak e Overlapping overlaps the peak labels All labels of all peaks of the mass list are shown e Tics only shows only tics on the top of detected peaks Orientation e Horizontal displays the labels of the m z values horizontally e Vertical displays the labels of the m z values vertically 2 4 10 10 3 Export Options ll Display Labels Export Calibration Numeric precision A Values E
65. ed in the spectrum and will be accepted If the width value is set too high small peaks will be regarded as noise flexAnalysis 3 0 User Manual Version 3 0 2 55 Graphical User Interface GUI Bruker Daltonik GmbH Height This value determines the upper part of the peak that is used to locate the peak position on the x axis i e m z If the separated peak area is symmetric the peak position will be found centric otherwise the maximum is shifted Edit Processing Method InternalCalibration ReadOnly Mass List a E F Find Mass List Find Edit B Processing Smoothing Baseline Subtraction Peak Detection Algorithm dom co co cm im ale ale ot Signal to Noize Threshold ot Relative Intensity Threshold Minimum Intensity Threshold Maximal Number of Peaks Peak Width Height Tena mi Baseline Subtraction Flatness mn lt MMedianLevel Figure 63 Method parameters dialog box Mass List Find Centroid Peak Detection Algorithm SNAP SNAP Sophisticated Numerical Annotation Procedure Figure 64 searches for known patterns in the measured spectrum and performs its own internal baseline correction and noise determination Therefore it is necessary to use SNAP together with raw data that have never been processed before i e no baseline subtraction and no smoothing With SNAP groups of peaks are detected in high throughput proteomics e g for identification of isotopic patterns and exa
66. ed whether the number of rings and double bonds present keeps within the bounds specified in Minimum and Maximum This will exclude all possible formulas with deviating double bond equivalence numbers Default is not selected The rings plus double bounds number is calculated from the number of atoms and their valency 2 2M X ValENCY jo 2 2 The valency of C and Si is 4 for N and P a valency of 3 is assumed For O and S it is 2 for H and the halogen atoms it is 1 For this limited set of atoms the equation simplifies to atom R db l R db Nic T 2 ME DF C1Br D Y 2 My py T l All the other elements are also considered Double bonds occurring with higher valence states like 4 or 6 for sulfur cannot be taken into account as these valence states are unknown Minimum Enter the minimum number of rings plus double bonds to be contained in a molecular formula at least This number may be negative for protonated clusters Default is 0 Maximum Enter the maximum number of rings plus double bonds to be contained in the molecular formula at most Default is O Apply nitrogen rule Check this option if the nitrogen rule should be applied This rule means that if the molecular ion M of an organic component has an even molecular weight it must contain an even number of nitrogen atoms And if the molecular weight is odd the 2 88 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical
67. election Undoes the Select All function e Load all selected spectra Loads and opens the spectra in flexAnalysis 2 4 1 3 Open Single Analysis In contrast to the Open command where one or more spectra can be opened at the same time the Open Single Analysis command opens only one spectrum Here it is necessary to browse to the fid file in the spectrum tree and select it flexAnalysis reads spectra which are saved in the so called XMASS file format In this file format raw data are stored in a file called fid Data already processed from flexAnalysis or flexControl are stored in a file called 1r If the analysis was not processed before the raw data file fid is loaded otherwise the result spectrum of the process 1r Additionally to the spectrum a method is loaded automatically A flexAnalysis method determines how to process an analysis The method that is loaded together with the analysis is either specified by the analysis itself due to method assignment during the save process in flexControl or if no specification has been done in flexControl or AutoXecute by the Default Method selection If a spectrum has never been saved with flexAnalysis before the method script part of the method is executed The drag and drop mechanism to open spectra works as follows a Start flexAnalysis b Start the Windows Explorer c Navigate for the fid file of the spectrum you want to open d Drag and drop the selected
68. er Manual Version 3 0 Bruker Daltonik GmbH 229 AXMANYSIS ISE VVINGOW dali 2 107 2 6 Mass Spectrum WINKOW coocccccocnnccccncoconncononocononnnonnnononnnonannnonannnnnnnnononnnnnnnnonananenas 2 108 Zot Mass IST VINCI Wa a a ite do Eos 2 108 2 8 Context Sensitive MENUS ooocccccconccccccoccnconoccnononcnnconnnonoononcnnononcnnnnnnnnnonanrnnonanennnnnns 2 108 2 8 1 Getting Context MenuS oocccconccncococcncccncononnnccnconncnnononcnnonnnrnnnnnrnnnonnnrnnonanennnnnas 2 109 2 8 2 Context Menus of the three flexAnalysis WiINdOWG ccccceesseeeeseeeeeeeeees 2 109 2 9 Pointers and Interactive Modes oooooccconncconcncccocnconocncnnnnnconcnnonanononcnnnnncnonnncncnnnnnnnns 2 110 A APPENDIX usina oia A l Alt Calibration Procedures dis A A 1 1 How to calibrate internally using the statistical peptide calibration A A 1 2 How to calibrate internally using the automatic assignment procedure A A 1 3 How to calibrate internally using the interactive assignment procedure A II A 2 High Precision Polynomal Calibration HPC oocooonncccccccnccccccnnccnononconononnnnnncononnanonos A III BAEZA Whats APC io io aR A III A 2 2 How can use HPC on the ultraflex ocoooccoccccnnoncccocococonnncocnannnnnonanonoos A III A 2 3 Can generate my own HPC methods with different instrument settings A IV A 2 4 How can check HPC performance occcco
69. erence between the two chosen peaks 2 46 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI Nh Phenylalanine 1 98 gt 5 Phenylalanine 533 056 533 056 304 041 304 041 l l j IMAN Y dele aih acacia Ld ee al 400 450 500 5 400 450 500 5 Figure 55 Annotation with and without Mass Difference 2 4 6 5 Preview Assignments The Preview Assignments command and the corresponding icon IMi of the toolbar offer a preview on all possible distances to higher and lower masses depending on the entered search tolerance that can be calculated for one starting peak together with the selected Building Block set An example is shown in Figure 56 To see the preview for one mass make sure that you have a peaklist activate the Annotation mode and activate the Preview Assignments button click on a peak in the spectrum display and keep the left mouse button pressed gt The matching Building Blocks will be shown You can choose one of these and release the mouse button to get this annotation 400 517 176 517 18 Tyr Cys _ s olu e e 88434 Phe llesleu ELA Trp 200 533 886 646 670 492 577 hn 489 184 r bhail 350 400 450 500 550 BOO 650 Figure 56 Example for possible assignments for the peak 517 18 Da flexAnalysis 3 0 User Manual Version 3 0 2 47 Graphical User Interface GUI Bruker Daltonik GmbH 2 4 6 6 Edit Building Blocks The Edit Buildin
70. estricted One way is to limit the allowed ranges of the occurring atoms The problem here is to set the limits in such a way that the correct combination will not be excluded A better way is to use a more intelligent algorithm to confine the number of combinations to be examined flexAnalysis uses an algorithm that is based on the fact that the integer part and the fractional part of the molecular mass are linearly independent for organic molecules up to a molecular mass of about 1000 This is used for combining C H N and O The other elements which usually occur in smaller numbers are dealt with using the straightforward try and error method The calculations in generating molecular formulas are based on the isotope masses and abundances and the valences given at the Table of Atomic and Isotopic Data we use The valences used for calculating the rings and double bonds the electron configuration and the nitrogen rule you can quickly inspect at the Table of Valences too Please refer to Appendix A 3 On selecting this command the Generate Molecular Formula dialog appears Figure 92 which allows for entering the m z value for the possible molecular formulas to calculate and defining how the calculation is carried out flexAnalysis 3 0 User Manual Version 3 0 2 85 Graphical User Interface GUI Bruker Daltonik GmbH Generate Molecular Formulas Measured m z 1000 000 Tolerance ppm 110 000 miz err ppm CS H6N130 17 999 99
71. ew peak list save the analysis It is not possible to remove background peaks without having identified them before To remove background masses automatically during an AutoXecute run a special flexAnalysis method is necessary Please contact the MALDI Software Support to get more information about this 2 4 3 10 Distance in Mass List s The macro Distance in Mass List s is used to search for specified fixed distances between peaks of one spectrum or of two spectra It may be used for instance to search for salt adducts in MS spectra amino acids in MS MS spectra or compare two ISD spectra lon Source Decay of the same sample but with different matrices in order to inspect differences in m z of the fragment series The spectrum or the spectra to be inspected have to be selected in the Analysis List Window At most two spectra can be selected In case of more selected spectra the macro will be disabled If not yet available a peak list from any of the two selected spectra has to be created Otherwise the message shown in Figure 37 appears flexAnalysis Mo peaklist available For spectrum autorun weri 6711115R eF Figure 37 No peak list available The Distance in Mass List s macro opens the dialog shown in Figure 38 flexAnalysis 3 0 User Manual Version 3 0 2 29 Graphical User Interface GUI Bruker Daltonik GmbH AutoDistance Distance ro Da Tolerance jo bm Co Spectrum aytorun_verifys1_G741 1SRef v Result
72. f the baseline but to minimize the distortion of the peaks TopHat should not be used for Protein spectra 2 4 7 6 2 FAST only for FAST Segment Spectra FASTW_K11 1 1046 5418 FAST FAST O_K11 1 1046 541 P X Process FAST Mass List Layout Display Options Perform Smoothing before Pasting Perform Baseline Subtraction before Pasting Figure 72 Method parameters dialog box FAST The FAST tab shown in Figure 72 is only available fora FAST Segment analysis This tab allows deciding if smoothing and baseline subtraction are performed before the FAST segments are pasted If both functions are selected first smoothing is performed The algorithms used for these processings are taken from the Process tab flexAnalysis 3 0 User Manual Version 3 0 2 63 Graphical User Interface GUI Bruker Daltonik GmbH 2 4 7 6 3 Recalibration Masses only for MS Spectra AAA A 4 Process Recalibration Masses Mass List Layout Display Calibration Strategy O Smart O Statistical Peptide Mass Control List PeptideCalibStandard mora v Name Ret Mass Da Bradvkinin M H _ mono Fo 3992 Angiotensin_ l M H mono 1046 5418 Angiotensin M4 H _mono 1296 6845 Substance_P_ M H _mono 1347 1354 Bombesin M H _mono 16198223 Renin_Substrate_ MmM H _mono 1758 9326 ACTH chp 17 M H _mona 2093 0562 ACTH_clip 18 39 M H _ mona 2465 1983 Sormatistatin Ah H 1 mann a4 4410 gt Peak Assignment Tolerance
73. field New License Key and press the Add button Bruker Daltonics LicenseManager New license key Existing licenses License key Walid Product name Unlimited Autosecute AunE ditor 3 0 unlimited BioT oole 3 0 unlimited BioTools 3 0 RapiDeNovo unlimited BioTools 3 1 unlimited BioTools 3 1 RapileNowo 06 01 06 ClnFroTools 2 0 Unlimited fles nalszis 2 4 Unlimited fles nalszis 3 0 unlimited flexLontral 2 4 unlimited tlesControl 3 0 ORSAI les radica 1 Figure 3 The Bruker Daltonics License Manager 1 4 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Introduction If the Bruker Compass Security Pack is installed licenses can only be added or removed by users who have the corresponding right In this case the dialog shown in Figure 4 appears to enter the password Re enter Your Password For security reasons please re enter your password here Figure 4 Enter the password to add or delete a license key A license key can be removed with the Delete button The warning shown in Figure 5 appears Bruker Daltonics LicenseManager Do vou really wank to delete the selected license key Some of your programs might not run or some of the options of these programs might not work without this license Figure 5 Warning before deleting a license The Bruker Daltonics LicenseManager dialog box can also be launched from the Compass Menu w
74. flexAnalysis User Manual A flexanalysis TOFUSER FLEXControl generated XMASS data c 2000 Bruker Daltonics File Edit Mass List Process Calibrate Method FAST Yiew Tools Window Help co Od Se le r ex a a i e mo gt re asa lo loca s a ea i xi Mass Spectrum E am 5 Angioll_PSD_1046 54_424 H MI 1SRef Raw Ala 25Ref Raw I 3SRef Raw A y 4SRef Raw A 5SRef Raw A y ESRef Raw A y 7SRef Raw Mln 8SRef Raw Mln ISRef Raw H Mla 10SRef Raw A lp 115 Ref Raw A y 125 Ref Raw Ml 13SRef Raw i Mhe 14SRef Raw E Mili Angioll_PSD_1046 54_424 n WI fast Raw 234 756 996 007 957 687 432 773 TH ig WET GEER RE 951 331 For Help press F1 ParametersOnly Parent mass 1046 540 Box Selection 4 BRUKER DALTONS Version 3 0 September 2006 Bruker Daltonik GmbH Copyright Copyright 2006 Bruker Daltonik GmbH This document is published by Bruker Daltonik Bremen All rights reserved Reproduction adaptation or translation without prior written permission is prohibited First Edition Printed in Germany PN 246503 The information contained in this document is subject to change without notice Modifications and additions may be made to the products described in this document at any time Bruker Daltonik Bremen make
75. g Blocks command is used to start the Bruker Daltonics BuildingBlockEditor application which allows you to view and edit the currently available building block sets to be used to automatically interpret m z differences Selecting this command starts the BuildingBlockEditor and loads the first of the alphabetically ordered building block sets shown in Building block sets The building blocks contained in this set are listed in Building Blocks along with their Full name Abbrev iation and chemical Formula Additionally the Symbol is shown which appears in the data window when the Annotation mode is active Various Bruker Default building block sets are available A lt BrukerDefault gt appendix to the building block set name indicates these You cannot alter or delete them However you can set up user specific building block sets by copying such a Bruker Default set to a new file name and then modifying the copy according to your own requirements or by creating a completely new building block set Bruker Daltonics BuildineBlockEditor File View Help Y Building block sets Amino Acids lt Bruker Default m Building blocks Full name Abbrey Symbol Formula Alanine Ala A C3H5NO Arginine Arg A C6H12N40 Asparagine Asn CAHEN 202 Aspartic Acid Asp C4H5NO3 Cysteine Cys C3H5NOS Glutamic Acid Glu C5HYNOS Glycine El C2H3N0 Histidine His CBH FM 30 _lso fLeucine llesLeu Iz C6H11N0 N E C Glutamine Gln E CS5HAN20 2 E G H Full na
76. gn working methods manually This failure is shown with the information lt Default Values gt in the status bar at the bottom of the GUI A method consists of two parts the method parameters section 2 4 7 6 and the method script section 2 4 7 7 The method script determines the sequence of processing procedures performed with your analysis while the method parameters configure each processing procedure Note The peaklist layout is also a method parameter If an analysis that has never been processed in flexAnalysis before is opened the method script is executed automatically and the Script in progress dialog is shown Figure 60 To get more information on the flexAnalysis method concept please refer to the Quickstart flexAnalysis Method Concenpt paf 2 4 7 2 Open The Open command and the corresponding icon i of the toolbar are used to open the Open flexAnalysis Method dialog Figure 59 to choose a new flexAnalysis method The new method replaces the previous one The name of the current processing method is displayed in the status bar of the GUI section 2 3 title bar of the Method Parameters dialog box Figure 61 that appears when selecting the Edit Parameters command from the Method menu as well as in the title bar of the 2 50 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI Method Script Editor Figure 77 and in the status bar of the GUI Here it is displayed a
77. h pointer This pointer is used to change the width of columns such as those in the Mass List Window It is displayed when the mouse pointer is positioned on the left or right border of a column head Pick pointer This pointer is used to directly select a mass spectrum in the Mass Spectrum Window It is displayed when the mouse pointer is positioned near a data point 3 pixel of a currently unselected mass spectrum Interaction Mode Mouse pointers Zoom In pointer This pointer is used to zoom the display range of a data window It is displayed when the Zoom In mode is active and when the mouse pointer is positioned on a data window s display Access Mouse pointer lt Shift gt key gt Zoom mode please refer to section 2 4 9 6 flexAnalysis 3 0 User Manual Version 3 0 2 111 Graphical User Interface GUI Bruker Daltonik GmbH Edit Mass List pointers Kel Ag These pointers are used to add a peak or to delete a peak from the Mass List in a spectrum displayed in the Mass Spectrum Window They are displayed in addition to the Data pointer when the Edit Mass List mode is active and the mouse pointer is positioned on the selected spectrum Which of both pointers is displayed at a time depends on the actual position of the Data pointer A A A The Delete pointer appears when the Data pointer is positioned on a data point that belongs to a mass peak already present in the spectrum s mass list A A T
78. he Add pointer appears when the Data pointer is positioned on a data point when its mass peak is not yet present in the Mass List window Access Mouse pointer lt Ctrl gt key gt Edit mode please refer to section 2 4 3 3 Internal Calibration pointer Rih This pointer is used to calibrate internally the mass spectra of an analysis It is used to manually select a peak from a mass spectrum displayed in the Mass Spectrum Window and to assign it to a selected reference mass Itis displayed in addition to the Data pointer when the Internal command from the Tools menu is active and the mouse pointer is positioned on the selected mass spectrum 2 112 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH A Appendix A 1 Calibration Procedures The following paragraphs summarize the three different recalibration procedures A 1 1 HOW TO CALIBRATE INTERNALLY USING THE STATISTICAL PEPTIDE CALIBRATION 1 Select exactly one spectrum in the Analysis List Window Make sure that this spectrum is displayed in the Mass List Window 2 Open the Internal Mass Spectrum Calibration dialog and select Statistical peptide if this option is not already selected See Golden Rules on page 2 42 Press the Calibrate button If satisfied with the new calibration press OK A 1 2 HOW TO CALIBRATE INTERNALLY USING THE AUTOMATIC ASSIGNMENT PROCEDURE 1 Select exactly one spectrum in the Analysis List Window Ma
79. he name of the user who has signed the spectrum e Meaning This column contains the comment that has been entered during signing e Valid This column contains remarks about the current validity status of data In this example the validity is not yet checked To do so apply the Check Validity button The attribute unknown will change to valid A double click on a signature in the list opens a dialog that shows if the state of all checked files is ok List of files that are part of this signature File State acqu Checksum OF acque Checksum mismatch analysis famethod Checksum OF fid Checksum OF pdata 1 1r Checksum mismatch pdata peaklist xml Checksum OF pdata proc Checksum miematch pdata procs Checksum OF splyoe Checksum OF Figure 17 List of files three file are mismatched 2 4 1 12 Export The Export command is used to export spectra and graphics into other applications or a peaklist to Excel To retain versatility spectra are exported in the common ASCII file format whereas graphics are exported in the common emf format Windows Enhanced Metafile With this macro only one Mass Spectrum can be exported With the Batch Processing macro and a suitable flexAnalysis method more than one mass spectrum can be exported at the same time On selecting the Export Mass Spectrum command the Export Mass Spectrum dialog appears to save a 2 column tabular with pairs of data points m z intensity
80. ical Peptide 22 1794 66355 Angiotensin_1 M H _mona 1296 68480 B a 1302 679994 24 1307 660973 25 1306 65137 26 1320 61162 2 1323 65426 20 1335 659085 Substance _P_ M H _mono 1347 73540 ei Za 1353 71997 Number of calibrants f Std dev ppd Before Last Fit In Last Fit Figure 49 Mass Spectrum Calibration dialog Calibration Strategy Smart If the option Smart Calibration is selected the calibration dialog looks as shown in Figure 49 SMART is an intelligent calibration strategy which significantly improves the identification success and the scores for PMFs Peptide Mass Fingerprints SMART calibration requires project specific mass control lists for calibration and background peak exclusion Such lists are generated with Bruker ProteinScape software or on individual customer request by the Bruker Software Support maldi sw support bdal de Common mass control lists are not suited for SMART calibration exception PAC_PeptideCalibStandard mcl 2 40 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI The SMART calibration is an iterative process which takes the following points into account the number of calibrants found in the respective spectrum the likelihood of these calibrants appearing the accuracy of the mass values as well as their distribution over the required calibration mass range The project dependent mass contr
81. ith the License command section 2 4 12 2 flexAnalysis 3 0 User Manual Version 3 0 1 5 Introduction Bruker Daltonik GmbH 1 5 Starting and Closing flexAnalysis 3 0 Start flexAnalysis 3 0 can be started via the Windows Start menu During installation of the Bruker Daltonics Applications package a Bruker Daltonics folder was automatically created in the Start menu s Programs folder which contains the applications delivered by Bruker Daltonik Select the flexAnalysis entry from this folder in order to start flexAnalysis 3 0 A second way to launch flexAnalysis 3 0 is double clicking the corresponding icon f IL on the Windows desktop Exit This procedure closes flexAnalysis 3 0 On closing the current user interface settings are automatically stored In order to exit flexAnalysis 3 0 click the application s Close button x or from the File menu select Exit or press Alt F4 If required answer the appearing confirmation request in order to save the current processing state of a modified analysis 1 6 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH 2 GRAPHICAL USER INTERFACE GUI flexAnalysis pepmix 20 06 2006 File Edit Mass List Process Calibrate Annotation Method FAST View Tools Window Compass Help sujan SF MAX TAMAR ie Ale AQ dn Pepmix_A24 3 Li MS u a a os ARE mae ilhi e i din E pepmixt0_424 1 122773 t li 15Ref 1308 980 1333 140 1347 827 1046 613 2093 204 13438
82. ke sure that this spectrum is displayed in the Mass List Window 2 Open the Internal Mass Spectrum Calibration dialog and select Interactive and choose a reference list containing the masses of your calibrants in the spectrum 3 Make sure that the settings for quadratic calibration mass tolerance and calibration list are feasible for your spectrum 4 Press the Automatic Assign button flexAnalysis 3 0 performs an assignment of calibrant masses and recalculates the calibration Co cz using this new assignment The line of assigned masses reference mass and mass of the spectrum is marked with a check in the calibrant list Additionally the error between both masses is displayed in ppm or in Da flexAnalysis 3 0 User Manual Version 3 0 A Appendix Bruker Daltonik GmbH A 13 HOW TO CALIBRATE INTERNALLY USING THE INTERACTIVE ASSIGNMENT PROCEDURE 1 Select exactly one spectrum in the Analysis List Window Make sure that this spectrum is displayed in the Mass List Window 2 Open the Internal Mass Spectrum Calibration dialog and select Interactive and choose a reference list containing the masses of your calibrants in the spectrum 3 Make sure that the setting for the zoom range fits to the quality of the current spectrum calibration and that the open calibration list is the correct one 4 Select the row of your calibrant in the reference mass list with a mouse click on that row The row is now m
83. l these data are displayed together with the experimental spectrum for comparison please refer to Bruker s PolyTools manual PN 205659 The PolyTools command is active if PolyTools is installed on the same computer After a spectrum is loaded in flexAnalysis this command is used to open the PolyTools program and to transfer the current spectrum 2 4 10 6 ProteinScape ProteinScape is a tool for storing organizing and analyzing data generated during the proteomics workflow The following basic tasks are supported e Data archiving and organization e Data viewing and reporting e In depth data handling e Data analysis ProteinScape is designed as a client server platform with a simple web browser based user interface It operates with industry standard relational database systems like Microsoft SQL Server The ProteinScape command is active if the PSClient is installed This has to be downloaded from the ProteinScape server The command ProteinScape is used to send spectra to the database ProteinScape For more detailed information refer to the manual that matches to the installed ProteinScape version 1 2 and 1 3 2 82 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI The communication of flexAnalysis with different ProteinScape versions works as follows e flexAnalysis 3 0 without psClient No communication with ProteinScape The command Proteinscape in the Tools Menu is disabled
84. lexAnalysis 3 0 2 2 Toolbar gt Sl BEX ELA WE Ao OP Oe E QQ 0 mes te Figure 8 Toolbar of flexAnalysis a ia JE Feature buttons have been given for commands that are regularly used These buttons are organized into several groups and can be found also in the menus 2 2 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI 2 3 Status Bar The Status Bar Figure 9 is located at the bottom of the GUI In the middle it contains the sample name of the currently active spectrum for a saved spectrum automsmsi0_1141 or the name of the loaded processing method PMF read only as long as the spectrum is not saved For Help press Fl aubomernsiO_ 1141 Annotate TOF Lser x 7110 0 w 176 00 For Help press Fl PME read only Annotate admin ES Ty 1 00 Figure 9 Examples of a Status Bar for a saved and un saved spectrum The second field displays the parent mass in case of fragment spectra not shown here The third field shows the selection mode Annotate which is just active The next field shows the name of the current user followed by two fields that show the current mouse pointer position in the Mass Spectrum Window 2 4 Menu Bar The Menu Bar Figure 10 consists of 13 pull down menus These menus contain all the features to operate flexAnalysis Open a menu with the left mouse button for example the File Menu as shown in the following section Most of the fe
85. lgorithm Carina Signal to Noise Threshold Figure 62 Processing Parameter Editor Mass List Find Any peak finder is optimized for a specific task In most cases if the elementary composition is known Figure 65 it is recommended to use SNAP for detection of groups of isotopic peaks Centroid is often used in case of protein spectra and benefits in defining mass accuracy The Sum Finder is used for time critical applications The following four parameters are the same for all algorithms Signal to Noise S N Threshold 2 54 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI The S N is defined as the height of the mass peak above its baseline relative to the standard deviation of the noise i e peak height 3 x o o standard deviation of the noise The higher the threshold the less peaks are found However if the selected value is too small noise may be regarded as peaks The recommended S N range varies from 3 1 to 10 1 In case of 10 1 you can be sure to have eliminated nearly all the noise Relative Intensity Threshold The Relative Intensity Threshold is a level in percent referring to the base peak which is the largest peak in the spectrum In cases where it is not clear whether the base peak refers to the sample or not the recommended value for this threshold is 0 zero Minimum Intensity Threshold A peak must have at least this intensity to be detected and annot
86. libration and sample spots have been acquired with the sequence e g on an Anchor chip target it is possible to redo the post processing in flexAnalysis in exactly the required order Browse Each Browse button opens a dialog to select a method for the respective spectrum type This method will be used for the batch process flexAnalysis 3 0 User Manual Version 3 0 2 33 Graphical User Interface GUI Bruker Daltonik GmbH e Start Batch Process Starts a batch process Figure 42 The current spectrum in progress will be shown in the appearing window The process for one spectrum can be stopped with the Cancel button _ Batch Process in Method Script in Progress Spectrum gt CCC y iy test 0 B5241 SR ets tid Method ExtemalCal Figure 42 Batch process in progress 2 4 4 6 Process Spectra The macro Process Spectra offers a simple method to re process MS spectra without the necessity of writing a script The dialog shown in Figure 43 contains the most important processing features They are the same as the corresponding in the different menus of flexAnalysis Process Spectra Undo All Processing Calibrate MS Spectrum Copy Calibration MS Spectrum Replace Calibration MS Spectrum Find Peaks _ Send to Protein cape Figure 43 Most important processing commands e Calibrate MS Spectrum refer to section 2 4 5 e Copy Calibration MS Spectrum refer to section 2
87. longs to the currently loaded method of the selected analysis 2 52 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI The running of the script is indicated by the dialog shown in Figure 60 Method Script in Progress gt lt Cancel Figure 60 Running method script The script processes the spectrum according to the particular processing operation s and parameters defined in the script and the parameter editor respectively To view a script before running the flexAnalysis Script Editor section 2 4 7 7 can be opened The script can then be started directly from here 2 4 7 6 Edit Parameters The Edit Parameters command the corresponding icon of the toolbar and the short cut Alt F2 are used to open the Parameter Editor According to the selected kind of spectrum MS LIFT and FAST in the Analysis List Window the editor offers a variable number of tabs with respective features Note All features contained in section 2 4 7 6 are method parameters If one parameter is changed and the currently active spectrum is saved the settings are not automatically available for other spectra If you want to use a special set of parameter for all your spectra you have to create a new method and save it with a new name 2 4 7 6 1 Process The Processing Method selected and adjusted here determines the results of all processings that affect the peaklist As long as the selected s
88. ly parameters or only the script to other analyses In this case the Paste Special command has to be used To test this feature load three spectra in flexAnalysis For the first one change some spectrum parameter section 2 4 7 6 and edit the script section 2 4 7 7 Mark the first spectrum in the treeview and choose Copy Now mark the other spectra lt ctrl gt key and select Paste or Paste Special to paste the method content Check the new settings for the two spectra in the parameter and the script editor Paste Special Source Unknown Source Paste Method Method Parameters Method Script Result Inserts the contents of the clipboard into your document as Method E Figure 25 Paste Special dialog 2 4 2 5 Delete The Delete command the corresponding icon A of the Toolbar and the short cut Del are context dependent flexAnalysis 3 0 User Manual Version 3 0 2 19 Graphical User Interface GUI Bruker Daltonik GmbH In the Treeview it is used to delete one or more processing steps corresponds to the Undo All Processing command but it does not delete the raw data file no feature exists in the flex programs that can delete the raw data In the Mass List Window the Delete command is used to delete the selected lines of the peaklist The command is not available for the Mass Spectrum Window 2 4 2 6 Select All The Select All command and the short cut Ctrl A are used to select the entire co
89. me Abbreviation Symbol Formula Asparagine Asn iN CAHENZ02 O Mia Monoisotopic mass 1140429274472 Average Mats 1141028041545 Save NUM Figure 57 Building blocks contained in the loaded building block set 2 48 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH 2 4 7 Method Menu Method FAST View Tools Window Cor Gale Open Save As P Run Script Fe EX Edit Parameters Alt F2 Ry Edit Script Ctrl Ez Select Default Figure 58 Graphical User Interface GUI Contents of the Method Menu The Method Menu Figure 58 is composed of various commands to configure method parameters 2 4 7 1 Overview Table 7 Feature button a El Features of the Method Menu Method Menu Shortcut Description commands Open Save Save As Run Script F2 Parameters Alt F2 Script Ctrl F2 Select Default Opens a processing method Saves the open processing method with the same file name if not write protected Saves the open processing method with a new file name Runs the Sax Basic script of the open processing method on the selected analysis Opens the method parameter editor Creates or edits the Sax Basic script that belongs to the open processing method Opens a dialog to select automatically attached default processing methods flexAnalysis 3 0 User Manual Version 3 0 2 49 Graphical User Interface GUI Bruker Daltonik GmbH Method
90. ment range and to recalibrate FAST segment spectra afterwards The operator can apply a new calibration file for recalibration in case of Segment Spectra acquired with flexControl versions lt 2 0 The Change Parameters command opens a dialog that lists the segment boundaries in m z Applying the radio button FASTLow FASTHigh the same segment boundaries are shown in the relation FASTLow FASTHigh flexAnalysis 3 0 User Manual Version 3 0 2 71 Graphical User Interface GUI Bruker Daltonik GmbH Change FAST Parameters Parent Mass 1046 54 mz Calibration File ACTH psd Segment Ranges in 5 FAST Low FAST High 2 Masses mz Y os E Jos E 108 105 o8 A hat A a ai a r A k A i al 1 08 el lao le lao lao ae iE erl Figure 82 Changing segment ranges using m z values The feature button in the right upper corner gt opens a dialog to load a FAST Segment Calibration File lt name gt psd This feature is only necessary and available if spectra acquired with flexControl versions lt 2 0 are used 2 4 8 4 Delete Segment Spectra The Delete Segment Spectra command is active if FAST segments are successfully pasted and the pasted spectrum has been saved The command closes all FAST segments and deletes them from the hard drive This feature corresponds to erasing a directory Ap
91. meters Process Recalibration Masses Mass List Layout Display Mass List ie ft mzl mz Signal to Noise SN Quality Factor Quality Fac Resolution Res Intensitpl Intens Areal rea Background Peak Flag Bk Peak Chi Square Chi 2 Full width at half masimum FY H b Relative Intensity Rel Intens Figure 75 Method parameters dialog box Layouts flexAnalysis offers 10 parameters that may be displayed in the Mass List Window The choice can be done via the Layout tab The layout is part of the flexAnalysis method So if you want to change it permanently for use with other spectra save the method afterwards If only the just opened analysis is saved the layout setting refers only to this analysis because it is saved in the file Analysis FAmethod that only belongs to this analysis e The pull down list can be opened with the button New Insert e The button Delete x is used to remove a peak property from the Mass List Window e The button Move Up ES or the related short cut Alt Up Arrow and the button Move Down or the related short cut A1t Down Arrow are used to change the arrangement of the displayed peak properties Note Some peak finding results are specific to a certain peak finding algorithm e g the Quality Factor If the parameter column is shown and the peaklist has been created for example with SNAP the Quality Factor column
92. n 3 0 Bruker Daltonik GmbH 2 4 10 1 Table 10 Feature button El hts 2 4 10 2 Overview Graphical User Interface GUI Features buttons of the Tools Menu Tools Menu commands Report Designer BioTools PolyTools ProteinScape Generate Molecular Formulas Macro Editor Customize Options Colors Glycopeptides Send Spectra to ProteinScape Description Opens the ReportDesigner Sends a spectrum to BioTools Opens PolyTools Opens Proteinscape Opens the molecular formula dialog Opens the macro editor Customizes menu bar and toolbar Customizes the spectrum display Coloring specific screen elements Opens the browser to select spectra to send to ProteinScape flexAnalysis 3 0 User Manual Version 3 0 2 79 Graphical User Interface GUI Bruker Daltonik GmbH 2 4 10 3 Report Designer The ReportDesigner command and the corresponding icon Cel of the Toolbar are used to start the Bruker Daltonics ReportDesigner which allows creating own report layouts Existing layouts can be modified and new ones can be set up To create a new report layout it is recommended not to modify the original layout but to save the modified layout to a new file name Otherwise it is possible that your modified layout may be overwritten by a later upgrade of the software Use in the ReportDesigner in the File menu the Open command to open and if needed edit an existing report layout or use the File men
93. n is calculated with current mass reference mass the error in ppm is calculated with erent Mass reference mass 10 reference mass Mass Control List Selection This pull down menu contains the available mass control lists MCLs Only lists with at least one mass marked as calibrant are offered The calibrants of the selected list are displayed in the box below Button Applying this button opens the Mass Control List Editor Figure 46 as also the button on the Recalibration Masses tab Figure 73 in the parameter Editor does section 2 4 7 6 3 MCLs can be changed and created with this editor For more information on how to use and create MassControlLists have a look in the Quickstart_MassControlLists_Compass_1 2 pdf available on the Compass for flex installation CD sl Mass Control List Editor Version 1 0 70 1 Method PeptideCalibStandard mono Read only Sele PeptideCalibStandard mona Peak label mz Tolerancelpp Calibrarit Background Adduct Calibrant frequency d Bradykinin 1 7 757 399160 AO E Angiotensin Il _ 1046 541800 al Angiotensin 1296 684800 AD Substance P_ 1347 735400 al Bombesin M H 1619 522300 AL Renin Substrat 1758 932610 AL ACTH_clip 1 1 2093 056200 50 ACTH_clip 18 3 2465 198300 AO Somatostatin 3147 47 1000 AD 4 aie al eis aie sl Ne M MEME Se MENEM Figure 46 Mass Control List Editor flexAnalysis 3 0 User Manual Version 3 0 2 37 Graphical User Interface GUI
94. nce can be measured very precisely even if the absolute calibration might not be optimized This value is especially useful to decide about the number of sulfur atoms dev Combined This value combines either dev MeanMass or dev m z for FTMS with dev IntensVarNorm For that purpose each value is normalized by its median value calculated for all formula candidates and the sum of the square roots is calculated This value therefore depends on the set of formulas The values are weighted in a data dependent way to optimize the selectivity of the resulting scoring value dev MeanMass Error RMS of intensity weighted mass differences between the measured masses and the theoretical masses for the given formula The formulas are sorted with increasing err ppm by default Clicking on the column header re arranges the list oppositely flexAnalysis 3 0 User Manual Version 3 0 2 91 Graphical User Interface GUI Bruker Daltonik GmbH Show Pattern 381 018 A 378 75 379 00 379 25 379 50 379 75 380 00 380 25 380 50 380 75 381 00 m z J rar N Overaid Stacked ALt SSS Figure 93 Comparing both existing and calculated mass The Show Pattern button allows you to compare the pattern of the measured mass and the pattern of the selected formula Figure 93 from the list of calculated masses Figure 92 Applying this button adds the pattern of the selected formula into the Mass spectrum Window and the formula itself into the Analysi
95. ncel the possibly done calibration If you want to apply additionally the original calibration constants use the command Apply raw calibration section 2 4 5 5 2 32 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI 2 4 4 5 Batch Process The Batch Process macro is used to re process spectra This command opens the dialog shown in Figure 41 Batch Process Spectra from vise D Adatapepmix 0_ 4244741 SAef id DO data pepmie0 424 241 SAef tid DO data pepmie 0 424 341 SA et tid MS Method LIFT Method FAST Method FAST Segment Method Start Batch Process Figure 41 Example of a Batch Process Dialog Folder Select data file s for re processing with the Open Multiple Spectra dialog The selected data path will be shown on top The complete path s are displayed in the list If spectra are loaded with the Folder button you have to select a method with one of the Browse buttons below and start the batch processing afterwards AutoX Run Alternatively open an AutoXecute run to load the data that have been acquired with this run If flexAnalysis methods are assigned via the RunEditor these methods will be taken for batch processing it is shown with the information from AutoXecute that appears after loading the run It is possible to change the methods selection in the run even if the data are already measured and have been processed If ca
96. nen E cats 2 8 2 4 1 7 SAVO ee a naan aaa E E ns eel aaa ae actunn eu 2 9 2 4 1 8 DANG AS lo 2 9 2 4 1 9 Save All A NS bianca vada 2 9 ZAAAO Sigm Electronic RECOM visi dade E E 2 10 ZAAEVA lt SMOW SIQNALINCS ai A 2 10 ZA AND EXPO iia do ee Mica ada ete thats Soi een esac 2 11 ZA Neto APIMEPTO VIE Wasi AA 2 12 ZA NA Plains 2 13 ZANE POPE ls Ii 2 14 ZA VO Elda caca 2 16 flexAnalysis 3 0 User Manual Version 3 0 jii Bruker Daltonik GmbH ZA E e e lo O 2 17 2 4 2 1 OVEA W eroana a seisce gas able inm ann cadeend A a 2 17 2 4 2 2 CODY paste siete setae be etic occ a E a te sed pi Le erasyce eet se del ye 2 18 2 4 2 3 A A a A 2 19 2 4 2 4 Paste SDCClal i soi2 a i a tad cola 2 19 2 4 2 5 DEIC CN ane ore a ol ld 2 19 2 4 2 6 o Ine a eo e Re eee eee nee 2 20 2 4 2 7 Select Nor a to ade da 2 20 2 4 2 8 WIGWOAIIAIY SIS talla da 2 20 2 4 2 9 VIEW SPECIAL 2 20 ZA ZNO SEA a e 2 21 ZA AIT Delal Colt e a a e de 2 21 2 4 2 12 Set Selection RANGE ccccoocccccconnncccononoconcnnconanonnnnannncononnnnonnnrnnonnannnnanoss 2 21 242 13 Clear Selecto in RR 2 22 Zee Ver BOX O CISCO arrai AR 2 22 Zar Mass SUMEN Ran 2 22 2 4 3 1 OV CIC dc aaa 2 23 2 4 3 2 A a E A van E aa ketene E A 2 23 2 4 3 3 oe 2 24 2 4 3 4 EDI dd 2 24 2 4 3 5 A cecal ited Sa cai ee ae aa 2 lasira ech a 2 25 2 4 3 6 MS MS Lista a eee wes 2 25 2 4 3 7 Edit Mass Control List died 2 27 2 4 3 8 Identify Background Peaks ccccococnocococcononoconncnnnnncoconcnnnonano
97. nnnnannnonnns 2 27 2 4 3 9 Remove BACKGFOUNG PCQKS uan 2 28 24 39 10 Distance in Mass Hist oda 2 29 244 Process MAN dat 2 31 2 4 4 1 A cabs enue sore cetties dacctecbicen eal ncn E uses 2 32 2 4 4 2 Smooth Mass pecto lid 2 32 2 4 4 3 Subtract Mass Spectrum Baseline occccocccccocncoccnnccnnnconanoconanonnanonos 2 32 2 4 4 4 UNdO All PROCESSING snn sche vee a a 2 32 2 4 4 5 o S noua a a a a a a auedaareunet 2 33 2 4 4 6 ROCESS SP CCI a A a e yidenceuatosastenasats 2 34 ZS Gallbrate Menz o cd 2 35 2 4 5 1 A A tea tagodaahdd esate adenaeet a cosarase 2 35 2 4 5 2 O 2 36 2 4 5 3 COpyGal Broto olaaa 2 42 2 4 5 4 Replace Callbra oia e a a 2 43 2 4 5 5 Apply Raw Call andas decana 2 43 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH 240 AMO non MM ai cien 2 44 2 4 6 1 OVEIVIOW sit bio 2 44 2 4 6 2 ANNO ni a a 2 45 2 4 6 3 A Rete enna oe Pn eee T AE en eee Mee nent Perera eee Or een 2 46 2 4 6 4 Show Mass Difference ooccccoonnccccocccnccnoccncnnoncnnnonanononnnnnnonnnrnnconanencnnanens 2 46 2 4 6 5 Preview ASSIGNOMeNES oooocccconcccconcnccnncncnncnonnncnnnncnnonnrnnnnnrnnnnnnnnonrnnonarinonoss 2 47 2 4 6 6 Edit Building BIOCKS sisirin eaa a a a 2 48 ZA Method MENU nierun a a ol 2 49 2 4 1 1 COVEN VICW ohyra A an ia eet 2 49 2 4 7 2 ODEN cerca et ao 2 50 2 4 7 3 e N APE AE F AA EEA E E nates senda shat ot a em lence a 2 52 2 4 1 4 DAVE Asia a e ete dde de 2 52 2 4 7 5 RUM OCDE seoa a o to td e de 2 52
98. nnotation Menu The Annotation Menu Figure 52 is composed of various commands that allow you to attach specific annotations to mass spectra to modify these annotations and to remove them again if you do not want them to be saved with the respective mass spectrum 2 4 6 1 Overview Table 6 Features of the Annotation Menu Feature Method Menu Shortcut Description button commands tT Annotate Alt Activates the Annotation mode ES Clear Deletes all annotations from the display and T from the Sequence List psp Show mass Hide display mass errors relative to theoretical differences mass differences Preview Spider web mode screen for all possible next Assignments peaks automatically en Edit Building et Blocks Opens the Building Block Editor An annotation is attached to one single point annotation or two difference annotation selected data points and is defined by its position in terms of the data coordinates intensity and mass Therefore when the display range is changed an annotation moves along with its associated mass spectrum Annotations can be attached to any peak of the peaklist 2 44 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI Each annotation consists of a value or text entry and a line marker that indicates the m z value s the respective annotation relates to Different annotation types are available depending on the kind of data you wish to annotate Ma
99. not replace a good quadratic calibration flexAnalysis 3 0 User Manual Version 3 0 A III Appendix Bruker Daltonik GmbH A 23 CAN GENERATE MY OWN HPC METHODS WITH DIFFERENT INSTRUMENT SETTINGS No HPC setup can only be done in the factory A 2 4 HOW CAN CHECK HPC PERFORMANCE Acquire mass spectra of samples with about 10 known peaks in the mass range 750 3500Da using the HPC method Proteomics HPC par The most relevant HPC correction takes place below 1kDa therefore it is essential that the sample contains calibrants between 750 and 1000Da We recommend to use the Bruker PeptideCalibration standard and to add Bradykinin fragment 1 7 757 Da in order to cover the low mass range Perform internal quadratic calibrations with flexAnalysis using the known 10 peaks as calibrants If HPC is switched on check box in flexAnalysis mean mass errors of about 3 5ppm are typically achieved whereas results are significantly worse about 10ppm with deactivated HPC A 2 5 CAN I USE HPC ON AN AUTOFLEX There is no need for HPC on autoflex because these instruments already have an almost perfect quadratic calibration curve A IV flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Appendix A 3 Table of Valences The valency of the atoms which are used for the calculation of the ring and double bonds the electron configuration and the nitrogen rule when using the Generate Formula command from the Tools Menu are as f
100. ntative spectra and find out the size SNAP average composition The drop down menu SNAP average composition allows selecting a composition Existing compositions can be modified compositions of other averaged substance classes can be added Modifications of Bruker defaults have to be saved to a different name flexAnalysis 3 0 User Manual Version 3 0 2 57 Graphical User Interface GUI Bruker Daltonik GmbH SNAF average composition AVveragine _ Fragment Peak Width OMSiloxan_ Na SILE Mass Difference s H I PEG_Na SILE Partner SH Threshold Po_Na Figure 65 List of SNAP average compositions For peptide applications a composition called Averagine is defined The conceptual meaning of the word Averagine Figure 66 and the term C 4 9384 N 1 3577 O 1 4773 S 0 0417 H 7 7583 is the averaged composition of molecules of the 21 amino acids corresponding to their abundance ratio This creation is denoted the first Building Block and is always located in the first row Figure 66 To edit a Building Block click B Edi MN Notepad E 154 File Edit Format View Help c201si 1H 6 Na 1 Figure 66 The pre installed average composition Averagine SNAP uses this average composition as often as it is necessary for approximation to the molecule under investigation lf a second composition is defined that is a mass existing only once in a monomer SNAP uses this one exactly once for mass determination The Second
101. ntent of the active window which is the Analysis List the Mass Spectrum or the Mass List 2 4 2 7 Select None The Select None command and the short cut Ctrl D refer to the active window and deselect everything that was previously selected 2 4 2 8 View Analysis The View Analysis command the short cut Ctrl Ins and disabling the root checkbox in the treeview are used to display or remove one or more selected analyses from the Mass Spectrum Display Analysis List Figure 26 Example for an analysis not displayed in the Mass Spectrum Window The analysis which is not displayed in the Spectrum Window remains in the Analysis List Window however the corresponding root checkbox is cleared Figure 26 2 4 2 9 View Spectrum The View Spectrum command and the short cut Ins are used to display or hide a selected spectrum or selected spectra in the Mass Spectrum Window 2 20 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI Pressing the lt Ins gt key a second time or selecting the related checkbox restores the display A spectrum is only displayed in the Mass Spectrum Window if both checkboxes for spectrum and dedicated analysis are selected Analysis List El w FP 0201 1 41 3 011 130 1 i C fast 1 t Figure 27 Tree view of a disabled not displayed spectrum 2 4 2 10 Set Color The Set Color command opens a palette of colors Figure 28 Figu
102. ocncccccnccccccoconncoconononnnncconononnnnnnonenenanos A IV A 2 5 Can Iuse HPC on an autoflex ooccccooccococonnonoconconcnnannnnnnannnconannnnonnnnnnos A IV Pes Table O Malone io ciacdss AV A E seaucacuaumavcaaencauscanceuunanus cynacsmeavmncetennneuecanie l I flexAnalysis 3 0 User Manual Version 3 0 vii Bruker Daltonik GmbH How to Obtain Support If you encounter problems with your system please contact a Bruker representative in your area or Bruker Daltonik GmbH hardware support Fahrenheitsir 4 Phone 49 421 2205 430 n Fax 49 421 2205 370 a Email maldi support bdal de http www bdal de software support Phone 49 421 2205 345 Fax 49 421 2205 390 Email maldisw suppont bdal de application support Phone 49 421 2205 184 Fax 49 421 2205 104 Email maldi appl support bdal de vili flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH 1 INTRODUCTION flexAnalysis 3 0 is a post processing software for spectra acquired with the Bruker Time Of Flight mass spectrometers of the flex series The program runs under Windows 2000 SP4 or XP SP2 operating systems Windows NT is not supported Before installing flexAnalysis 3 0 for the first time the program Microsoft NET Framework 1 1 must be installed because flexAnalysis since the release 2 2 is based on features which Microsoft NET Framework 1 1 provides This program is also part of the Compass installation CD Figure
103. oggles Mass Spectrum Window between Range Selection and Box Selection modes The Copy command the corresponding icon of the Toolbar and the short cut Ctrl C are used to copy context dependent data into the clipboard Afterwards the copied information may be pasted to other spectra or other applications like Microsoft Office programs According to the active flexAnalysis window the user can use Copy in the e Analysis List Window treeview to copy the method parameter and script from the selected spectrum e Mass Spectrum Window to copy the currently shown graphic independent of the spectrum that is possibly selected in the treeview e Mass List Window to copy the currently marked line s of the peaklist 2 18 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI 2 4 2 3 Paste The Paste command the corresponding icon E of the Toolbar and the short cut Ctrl V are only available for the Analysis List Window treeview With this button the just copied method parameter and script can be pasted to one or more other analyses This feature is helpful if you have adjusted and saved a new method and want to apply it to several other spectra 2 4 2 4 Paste Special The Paste Special command is also only available for the Analysis List View and applies the method which has been copied before Since a method consists of parameters and a script the operator may want to transfer on
104. ointers and Interactive Modes To facilitate operating with flexAnalysis different pointers are provided anyone associated with a specific function Data pointer Vv The data pointer accompanies vis a vis the arrow pointer of the mouse within the Mass Spectrum Window In the case of two or more spectra the data pointer resides on the last generated spectrum The m z value and the related intensity of the current position of the data pointer are displayed in the Status Bar of the Graphical User Interface at the right Pointers for direct manipulation Horizontal Window Border pointer docked windows Y undocked windows This pointer is used to change the height of a window It is displayed when the mouse pointer is positioned on the upper or lower border of a window Vertical Window Border pointer F docked windows undocked windows This pointer is used to change the width of a window It is displayed when the mouse pointer is positioned on the left or right border of a window Horizontal Scaling pointer ees This pointer appears below the abscissa of the Mass Spectrum Window It is used to shift the spectrum and the scale horizontally 2 110 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI Vertical Scaling pointer Left This pointer appears below the ordinate of the Mass Spectrum Window It is used to shift the spectrum and the scale vertically Column Widt
105. ol lists do not only include the peptides whose sequences and theoretical masses are known e g trypsin autolysis products and keratin fragments but normally a large number of background peaks Their sequence is not known and their masses are determined by the average obtained from a large number of well calibrated spectra obtained during such a project Such background peaks are project specific and are very dependant on sample preparation digestion method staining method and preparation Statistical Peptide The third calibration strategy Statistical Peptide is only useful when peptide spectra are calibrated Internal Mass Spectrum Calibration Ed Calibration strategy eras Interactive Smart RStatistical Peptide 9 Peak assignment tolerance Default 400 ppm User defined s00 ppm Number of calibrants Std dey opm Before Last Fit In Last Fit E Figure 50 Mass Spectrum Calibration dialog Calibration Strategy Statistical Statistical Peptide is a so called self calibration of TOF peptide spectra It is a powerful tool especially in cases where no internal calibrants are Known Note A statistical calibration needs no calibrant list This strategy is based on the fact that the monoisotopic masses of peptides are distributed in a very regular way at least in the mass range which is used for protein flexAnalysis 3 0 User Manual Version 3 0 2 41 Graphical User Interface GUI B
106. ollows the atoms are ordered with increasing atomic number atomic weight flexAnalysis 3 0 User Manual Version 3 0 A V Appendix Bruker Daltonik GmbH e ao E a h m a T S RS E T 1 2 Pr 3 Ra 2 P E BEE a Ac ti too 4 Pm 3 m _ J4 As J3 Sm 2 NO i Y T slol D gs alrici lt alm rolam epr Cc U jo 3 Cp eae a tT o O Z O m JJ C gt O 73 Cc O K B Gd Tb Dy Ho lu a Ta Re CN e ASE Ao 3 ET AE A wm i i NO P 2 Bh o Hs o 5 2 m Jo 2 qn J3 Ds o N 0 w N i i Cc Cc lt lt gt op gt D S O D SS aq oO BES In le i C Cc oO 7 i i E O O o At ji uh i Table of Valences A VI flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Index A About Command 2 107 Analysis List command 2 101 Analysis Window 2 108 Assistance viii 2 107 Auto Scaling command 2 74 B Batch Process command 2 33 BioTools command 2 81 Box Selection command 2 22 C Change Parameters command 2 71 Clear command 2 24 2 25 Clear Range Selection command 2 22 Close command 2 8 Color command 2 21 Colors command 2 100 Compass Security Pack 1 1 Contents lil Copy Calibration command 2 42 2 43 Copy command 2 18 Customize command 2 95 D Default Color command 2 21 Delete command 2 19 Delete Segment Spectra command 2 72 Distance in Masses command 2 29 flexAnalysis 3 0 User Manual Version 3 0
107. on Smoothing flexAnalysis supports three different smoothing algorithms Savitzky Golay Gauss and Chemical Noise Which one is used depends on the application For all filters the smoothing Width can be chosen The Width range is from O to 100 the user is asked to find out the best fitting value The Cycles determines the number of smoothing processes on one analysis One cycle is recommended This parameter is not applicable together with the algorithm Chemical Noise The idea of the Savitzky Golay algorithm is to calculate polynomials in the neighborhood of each data point to get a smoothing of the data This can be formulated as M Vi Le Cire k M with coefficients c The parameter M in the formula is calculated from the given m z smoothing width The number of smoothing cycles can also be chosen which gives the option to apply this smoothing filter multiple times e Edit Processing Method pepmix 0_A 4 1 Mass List Find omoothing Edit B Processing Smoothing Baseline Subtraction APEL SavitzkyGolay a Width 10 2 mz Cycles 1 Figure 70 Method parameters Smoothing Processing Baseline Subtraction flexAnalysis 3 0 User Manual Version 3 0 2 61 Graphical User Interface GUI Bruker Daltonik GmbH The settings for Baseline Subtraction processes can be adjusted in the section Baseline Subtraction section 2 4 4 3 Three different algorithms are offered ConvexHullV3 Me
108. on average molecular weight 1 2 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Introduction 1 1 Getting Started In case of installing flexAnalysis 3 0 as a standalone version it can be installed from the Compass 1 2 installation CD Figure 1 1 2 Installing flexAnalysis 3 0 Please refer to the separate installation instructions on the Compass installation CD or contact Bruker via e mail page 1 1 1 3 Removing flexAnalysis 3 0 1 oS p p e w 1 Start your Windows application Click MA Point on Settings Click Control Panel Double click Add Remove Programs From the applications list select Bruker Daltonics flexAnalysis 3 0 Click Add Remove Follow the Install Shield Wizard instructions to remove flexAnalysis 3 0 from your system flexAnalysis 3 0 User Manual Version 3 0 1 3 Introduction Bruker Daltonik GmbH 1 4 Licensing flexAnalysis 3 0 Before flexAnalysis starts for the first time the dialog shown in Figure 2 appears flexAnalysis Bruker Daltonics Flex Analysis has not been licensed yet In order to use Flex Analysis the application must be licensed First Press OK to license Flex Analysis now Press Cancel to terminate Flex Analysis Figure 2 FA is not yet licensed After applying the OK button the Bruker Daltonics LicenseManager Figure 3 appears The flexAnalysis license key comes in a separate envelope with the software Enter the key in the edit
109. pear w Bradykininf1 7 M 757 39916 757 42714 12 0 00875 angiotensin _II_ M 1046 54180 1046 61258 9 0 00972 Zoom Range off w w Angiotensin_I M 1296 684850 1296 76864 3 0 00425 w Substance P M 1347 79540 1347 82697 6 o oogs0 e e eae eit aire s Bombesin M H H 1619 862230 1619 91957 2 0 00255 From Mass Control List Renin Substrate 1758 93261 2 Near Neighbor 10 ppm w ACTH clip 1 17 M 2093 086620 2093 20438 2 0 00375 User Defined 500 ppm w ACTH clipf1s 39 2465 19830 2465 30791 10 0 02355 w Somatostatint2s 3147 471100 3147 62753 5 0 01612 Calibration Fit Order Linear 2 Quadratic Number of calibrants Mass Spectrum Calibration dialog Interactive 5 Std dey ppm Before Last Fit 61 In Last Fit 7 Coronas Figure 45 Calibration Strategy flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI Interactive Calibration The calibrant list shows names and masses of known substances the corresponding masses found in the currently active spectrum and the error in ppm and in Dalton Interactive Calibration can be done automatically with the button or manually with click in the list on one calibrant followed by a click in the spectrum left to the corresponding peak Masses of the peaklist are then filled in the calibration list and the error is re calculated with each new assignment The error in Dalto
110. pectrum has not been saved the name of the chosen Processing Method is shown in the dialog After the spectrum is saved the sample name is shown analog to the method name sample name shown in the status bar before and after the spectrum is saved flexAnalysis 3 0 User Manual Version 3 0 2 53 Graphical User Interface GUI Bruker Daltonik GmbH CalibratePeptideStandards pepmix 0_A24 1 Method Par 2 X Process Recalibration Masses Mass List Layout Display Processing Method IntermalCallbration Figure 61 Process tab Since Compass 1 2 for flex i e flexAnalysis and flexControl 3 0 the peak detection algorithms as well as processing settings like baseline subtraction and smoothing are defined in Processing Methods prp that are edited via the Processing Parameter Fditor Figure 62 These parameter containers can be created edited and used from flexAnalysis flexControl and AutoXecute Detailed information can be found in the corresponding Quickstart_ProcessingParameterMethods pdf Mass List Find The flex programs support three different detection algorithms Centroid SNAP and Sum These are used to detect mass peaks calculate the corresponding m z values and many other peak parameters such as intensity or resolution re Edit Processing Method default ReadOnly E Mass List Find Mass List Find Edit B Processing Smoothing Baseline Subtraction Peak Detection A
111. plying this command activates message shown in Figure 83 Afterwards only the pasted FAST spectrum remains on disk Confirm Folder Delete al Are vou sure you want to remove the Folder 1046 5418 FA5T and tel move all its contents to the Recycle Bin Figure 83 Message before erasing the folder 2 12 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI 2 4 9 View Menu View Tools Window Compass Help lt Overlaid Stacked List Auto Scaling Alk Fo Scale to Entire Spectrum Chrl F9 4 Horizontal 4uto Scaling gt Vertical Auto Scaling Set Display Range CA 200m F3 CQ Zoom Shift F9 a Undo Zooming Alt LeFt Toolbars Status Bar Figure 84 Contents of the View Menu 2 4 9 1 Overview Table 9 Features of the View Menu Feature View Menu Shortcut Description button commands Toggles the abscissa and if possible the Auto Scaling Alt F9 ordinate of the active data window between auto scaling and manual scaling All currently displayed spectra are completely SEO Ente Ctrl F9 shown however auto scaling is not switched Spectrum on Horizontal Returns from manual horizontal scaling to Auto Scaling auto scaling El Vertical Auto Returns from manual vertical scaling to auto E Scaling scaling flexAnalysis 3 0 User Manual Version 3 0 2 73 Graphical User Interface GUI Bruker Daltonik GmbH Feature View Menu Shortcut Description button comm
112. pointer iN is displayed when the mouse is placed near to a peak that currently has no annotation whereas the A delete pointer AZ is placed next to an already detected peak in the spectrum If the new peak can be found using the Mass List Edit Method Parameter settings the results are added to the peak list and a label appears in the display To remove a peak from the peak list place the Delete pointer to the peak and press the left mouse button The Edit mode becomes also active when the mouse is inside the Mass Spectrum Window and the lt Ctrl gt key is simultaneously pressed 2 4 3 4 Edit Direct The Edit Direct command the corresponding button A and the short cut Ctrl Alt are used to add an annotation manually This pair of m z intensity data can be located at any position in the spectrum the x position is independent of a data point If this mode is activated the mouse in the display will get a crosshair to better see the mass and intensity values Additionally you will get a tooltip which shows these information and the spectrum name as it is shown in Figure 31 This feature will be deactivated right after you have added an annotation manually 2 24 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI tae 2 BS Sh Bh tA ee E PH Yq a GG ce fete Mass Spectrum mz Sh Res Intens P A Tor 304 114 5 alar 685 66 1000 536 22 9 4566 153 72 1001 548 101 7 3044
113. r detailed information on the Mass Control List Editor refer to the Quickstart_ MassControlLists Compass1 2 doc 2 4 3 8 Identify Background Peaks The Identify Background Peaks command is used to identify background peaks in a spectrum on the basis of a Mass Control Lists mcl that has to be selected with this command To create or modify an existing background list it has to be opened with the Mass Control List editor section 2 4 3 7 Only masses that are marked as backgrounds in this list Figure 34 can be used to identify background masses in a spectrum Masses can be marked as calibrants and or as backgrounds The Tolerance value is valid for both sl Mass Control List Editor Version 1 0 71 0 Method background Read Peak label m_z Tolerance pp Calibrant Background matris 568 13 100 a iw T 659 38 100 E iw matris a55 1 100 i iw matrix 1060 1 100 x we T 163 057 100 La iw T 2273 16 100 i g Figure 34 Example of a background mass list flexAnalysis 3 0 User Manual Version 3 0 2 27 Graphical User Interface GUI Bruker Daltonik GmbH After a Background list has been applied to a spectrum all peaks are marked with true or false in the column Bk Peak that can be selected as additionally shown column in the peaklist An example is shown in Figure 35 The column Bk Peak has been added to the Mass List Layout Mass List Layout tab in the Method Editor section 2 4 7 6 4 Peaks which are identified as backgro
114. re 28 Palette of provided colors This command is used to change the color of one or more selected mass spectra If only one spectrum is selected a white frame indicates the current color used for the selected mass spectrum To change the color simply click the desired color box 2 4 2 11 Default Color In flexAnalysis color assignments are performed according to a color hierarchy Each color in this hierarchy has a unique value Colors with higher values will be used prior to colors with lower values The Default Color command assigns colors to the selected spectra which are currently unused and have the highest scores in the color hierarchy 2 4 2 12 Set Selection Range The Set Selection Range command is used to select a mass range of the currently active spectrum within the Mass Spectrum Window for example to run a peak picking process in this specific range Additionally an intensity range can be defined when the button Range box selection type is pressed In the dialog box minimal and maximal mass and intensity values can be entered to define the ranges If only the mass range is requested choose Range selection to disable the intensity range flexAnalysis 3 0 User Manual Version 3 0 2 21 Graphical User Interface GUI Bruker Daltonik GmbH Range Selection Mass m z 1000 2000 Intensity 100 MEL ih Range box selection type Figure 29 Range Selection Dialog box 2 4 2 13 Clear Selection This comm
115. remains empty 2 66 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI 2 4 7 6 5 Display pepmix 0_A24 1 pepmix 0_A24 1 Method Parameters AA Process Recalibration Masses Mass List Layout Display Displayed numeric precision Mass precision 3 Result Display Options Replace Original Figure 76 Method parameters dialog box Display In this dialog the number of displayed digits for masses in the display and the Mass List Window peaklist can be changed Checkbox Replace Original When this box is activated default setting the new result spectrum after baseline subtraction or smoothing replaces the previous original spectrum in the Analysis List Window The spectrum color remains the same No additional spectrum is displayed If this box is not selected an additional spectrum is attached to the treeview with different color due to the color hierarchy 2 4 7 7 Edit Script The Edit Script command and the short cut Ctrl F2 are used to open the flexAnalysis Sax Basic Script Editor This is an interactive design environment to develop test and execute Sax Basic scripts Sax Basic is Visual Basic for Applications Y compatible Scripts are used to process analyses automatically In the flexAnalysis environment scripts are part of an analysis or a method The contents of a script are normally processing instructions for a particular analysis the me
116. ruker Daltonik GmbH digest measurements The masses accumulate around masses Mmean at a distance of approximately 1 00048 Da The masses Mmean are used as calibrants for all peaks in the mass list in a statistical calculation of the new calibration constants Golden rules for statistical calibrations Statistical calibrations are applicable for peptides only Under good conditions a mass error of 20 40 ppm can be reached The more peaks are available in the mass list and the better is the distribution of these peaks over the mass range of interest the better is the result of the statistical calibration As a rule of thumb about 20 peptide peaks are needed to obtain a meaningful result The maximum mass error of the peaks must not exceed about 0 4 0 5 Da 500 ppm at 900 Da At about 0 5 Da the algorithm cannot decide which mass is concerned This can lead to an incorrect calibration of one Da If your feel certain that the external calibration of the given spectrum is significantly better than 500 ppm you can reduce the peak assignment tolerance accordingly This can slightly enhance the performance of the statistical calibration Strong matrix cluster peaks between 700 and 1500 Da can affect the quality of the Statistical calibration results This happens because the stoichiometric composition of matrix clusters and peptides are strongly different 2 4 5 3 Copy Calibration With this command the calibration constants of a spectrum c
117. s List Window as a subentry of the original spectrum Note flexAnalysis does not save these processes This feature is for viewing only however and may be printed 2 92 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI Save Results Allows you to save the molecular formula results list in a xml file Opens the Save As dialog Report The Report button opens a HTML page containing the key information of the Generate Molecular Formulas operation Figure 94 3 Generated Molecular Formulas Microsoft Internet Explorer File Edit View Favorites Tools Help de Back mb ie at 0 Search Li Favorites eMedia ca yr Sh i BE Address 7 21 Documents and Settings MNeumanniLocal Settings Tempi MFE 4 html Generated Molecular Formulas Formula Mass Error DelEqg N rule Electron Ic C sa H 3H 36 926 4075 0 0592 32 00 odd Ic S3H44N 2905 926 4075 0 0649 26 50 ever Ic i4 H 50 N 220 10 926 4075 0 0706 21 00 odd Ic 35H 56 N 15 0 15 926 4075 0 0762 15 50 ok even Ic S6H62N 60 20 926 4075 0 0819 10 00 odd e Cahn beh IZ 926 4075 0 0876 4 50 a even Ic C50 H60N 30 14 926 4070 0 4662 22 20 ever Ic 45 H54N 100 926 4070 0 4719 20 00 odd Ic 48 H48N 1704 926 4070 DAI 53 50 ok even Ic 17424 N45 0 1 926 4060 0 6017 25 00 odd Ic IBH40N 4106 926 4080 0 6073 19 50 ok ever Ic 1
118. s are central elements in flexAnalysis They determine the processing of the analysis An analysis is always associated with one method This assignment takes place during the load process and depends on several criteria First of all it depends on whether the analysis has already been processed and saved with flexAnalysis or not If the analysis has once been saved with flexAnalysis a file called analysis FAMethod was created next to the fid file in the spectrum tree that contains all method settings parameter and script that were active at saving time This file is loaded when the analysis is opened again In this case the spectrum sample name is shown in the status bar at the bottom of the GUI If the analysis has never been saved with flexAnalysis before the program checks whether the acquisition program flexControl Save As dialog or AutoXecute Processing tab has specified a flexAnalysis method after the acquisition In this case this method is assigned If the assignment fails either because the method is broken or does not exist the Default Method see 2 4 7 8 is loaded since it is not possible to open a spectrum without a method In all these cases the name of the method is shown in the status bar at the bottom of the GUI at least as long as the script is running In some cases the assignment of the Default Method may fail too Then so called Default Values and an empty script are loaded to have access to the data and to assi
119. s long as the analysis has not been saved Open flexAnalysis Method Look in gt Hex nalyzishdethods v m CalibratePeptideStandards FAMSMethod m CalibratePeptide Statistic FAMSMethod F ExternalCalibration FAMSMethod m ExternalCalibrationPS PAMsMmMethod Parametersonly FAMSMethod 2 PMF FAMSMethod 2 PMF_PS FAMSMethod 2 Proteins 5 20kDa FAMSMethod Gorm 5 ee My Network Files of type MS Method Figure 59 Open flexAnalysis Method dialog box How to assign a method There are different ways to assign a new method to a selected analysis This may be required if it is necessary to use a method that is different to the current method settings Assigning a method with the open command 1 Select the analysis that shall obtain a new method 2 Click le or 1 From the Method menu select Open 2 Select the method you want to attach 3 Click Open Attaching a processing method of another analysis with copy and paste section 2 4 2 flexAnalysis 3 0 User Manual Version 3 0 2 51 Graphical User Interface GUI Bruker Daltonik GmbH It is not required that this method exists in the flexAnalysis methods directory for a saved spectrum the method Analysis FAMethod from the spectrum tree is used 1 Select the analysis whose method you want to attach in the tree view 2 Right click the selection and select Copy or Click Es 3 Select the analysis that shall obtain a new method 4
120. s no warranty of any kind with regard to this material including but not limited to the implied warranties of merchantability and fitness for a particular purpose Bruker Daltonik Bremen shall not be liable for errors contained herein or for identical or consequential damages in connection with the furnishing performance or use of this material Bruker Daltonik Bremen assumes no responsibility for the use or reliability of its software on equipment that is not furnished by Bruker The names of actual companies and products mentioned herein may be the trademarks of their respective owners li flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Contents T INTRODUCTION vasta ala caia tand 1 1 E E A a 1 3 12 ollaStallitnG Te xXANAlVSS 3 Ossa A 1 3 1 3 Removing flexAnalysis aaa 1 3 1 4 Licensing flexAnalysis 3 Da calas 1 4 1 5 Starting and Closing flexAnalysiS 3 0 ooooocccccnoncccccncccnconononnononnonoonaronnnnncnnononnnncnnaninnos 1 6 2 GRAPHICAL USER INTERFACE GUJ c ooooocccconnncccoccccoconcccocnnconoccnnorononnconnnnanonnnannronnananennas 2 1 Zils Te a a oi Ata 2 2 ZL VOD io a maniac swans ts 2 2 ZS E sere ace ss ese acta ae tame ees ee saan ce ae tame E 2 3 2 MED tai aid doo coda 2 3 A ee een een e te ener ere pe ete Ree mince a Cente Nery ene eee 2 4 2 4 1 1 A 2 4 2 4 1 2 81 AAE E A ee ee ea eee eee 2 6 2 4 1 3 pens male AMA SIS cido E 2 17 2 4 1 4 A A 2 8 2 4 1 5 ios o ads 2 8 2 4 1 6 Edad and Un IO din tie
121. se the same UserManagement server In this case it is necessary to un lock only one program A locked program can only be un locked from the user who locked it or from the UserManagement administrator The command is only available in case of an installed Bruker Daltonics UserManagement 2 104 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI 2 4 12 5 Compass Desktop The Compass Desktop command opens the so called Bruker Compass This GUI offers access to the installed Bruker programs distributed in Acquisition Processing Data Interpretation and the belonging manuals 2 Bruker Compass 1 2 AE File Options View Help Wf e Acquisition e Processing N e Data A e BioTools on e Hal sx GenoTools YZ Yl a BRUKER DA TONIOS NUM 16 10 12 Figure 107 Compass Desktop GUI 2 4 12 6 FlexControl The FlexControl command is used to open flexControl or bring it to front if it is already open Data transfer cannot be achieved with this command or with any other 2 4 12 7 BioTools The BioTools command is used to open BioTools or bring it to front if it is already open Data transfer cannot be achieved with this command i e no spectrum is sent to BioTools To send spectra use the BioTools command from the Tools menu section 2 4 10 4 flexAnalysis 3 0 User Manual Version 3 0 2 105 Graphical User Interface GUI Bruker Daltonik GmbH 2
122. sequence that was loaded in flexControl AutoXecute tab during manual or automatic MS data acquisition If no sequence was loaded during the data acquisition the button is disabled With this feature it is possible to do automatic LIFT measurement with masses that have been measured and selected manually in the AutoXecute sequence no MS measurement has to be specified The AutoXecute method and the flexAnalysis method necessary for automatic MS MS acquisition and post processing are added automatically to the sequence due to settings performed in the flexConfigurator For more information on the method assignment please have a look in the flexControl user manual 2 26 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI It is possible to add peaks from different spectra to the list And as seen in Figure 32 these spectra may have been acquired on different sample spots The only restriction is that they must have been acquired on the same target Otherwise the message shown in Figure 33 appears during spectra selection flexAnalysis You are selecting an analysis From a different target The MS MS list can hold peaks From a single target only Do vou Wish bo postpone clearing the M5 M5 list This will allow you to append the peaks to the Autoxecute Run or send them to FlexControl respectively Figure 33 Information concerning different targets 2 4 3 7 Edit Mass Control List Fo
123. sic script 2 4 7 8 Select Default Every spectrum that is opened in flexAnalysis needs to have a flexAnalysis method This method is automatically assigned on different ways depending on save processes in flexControl AutoXecute or flexAnalysis that occur before so the user is not requested to do anything During the manual saving in flexControl or during AutoXecute runs it is possible and sometimes recommended to indicate flexAnalysis methods that are used for post processing later on If no method has been indicated each spectrum automatically gets a default method assigned during the load process This default method is determined in the dialog Default Method Selection For any of the four possible spectra types suitable methods are available Figure 78 Default Method Selection Default Method for MS Spectra PMF MS MS FAST Segment Spectra ParametersOnly MS MS FAST Spectra ParametersO il MS MS Lift Spectra SNAP_full_process Figure 78 Default methods that are assigned to spectra that have no method 2 68 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI The methods ParametersOnly contain a set of suitable parameters and an empty script For this reason empty script gt no commands no processing and especially no saving takes place if a spectrum gets ParametersOnly assigned The pre installed default settings are PMF for MS spectra and SNAP full pro
124. ss spectra peaks can be annotated with their corresponding m z values of m z difference building block information or text 2 4 6 2 Annotate The Annotate command the corresponding icon 11 of the toolbar and the shortcut Alt are used to activate the Annotation mode The toolbar is expanded with the Annotation features shown in Figure 53 Amino Acids TOO ppm v vaine Flame rey itt r Figure 53 Annotation toolbar These toolbar features are pre installed some others concerning the annotation font and orientation can be additionally selected Customize Toolbar section 2 4 10 9 Amino Acids Y This Drop down box offers all available Building Block Sets the sets can be edited with the Building Block editor Select a set from the list that possibly contains building blocks that match to the currently selected spectrum oom The Building Block Search Tolerance has to be entered manually it is possible to change between Dalton and ppm The Tolerance is used for the preview assignment feature section 2 4 6 5 Valine wt The field Annotation Text can either be filled manually with any text that is then transferred into the display but it is also filled automatically with the Building Block information that belongs to the Annotation that is just activated in the display mouse click Depending on the selection of the Abbreviation Box see below the full name symbol or abbreviation of the Building Block is shown
125. tatistical peptide calibration procedure A l Statusbar 2 3 Statusbar command 2 78 Subtract Mass Spectrum Baseline command 2 32 T Title bar 2 2 Toolbar 2 3 Toolbars command 2 17 tools menu 2 35 U Undo All Processing command 2 32 Undo Zooming command 2 76 User management 1 1 V View Analysis command 2 20 View Spectrum command 2 20 W Window menu 2 101 Z Zoom command 2 75 Zoom X command 2 76 flexAnalysis 3 0 User Manual Version 3 0
126. ter and special report layouts can be chosen here The button Preview at the bottom of the dialog opens the actual preview and displays the selected spectrum as it would appear in the printout Layouts can be created designed and modified with the Report Designer The button Report Designer starts this application It is described in section 2 4 10 2 and in the Report Designer manual on the installation CD 2 12 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI Print Preview Printer Mame HF Lazenlet 6P 6MP PostScript v Status Read Type HP Laseret 6P BMmP PostScript Where pevhb eptd Comment Direct printing recommended for PostScript and PDF drivers 2 Use Printer resolution C Print with 300 DPI Print range Copies all Number of copies 1 alf Report layout Layout LIFT portrait v Orientation Portrait Landscape Preview Cancel Figure 19 Print Preview dialog box If own report layouts are created on the basis of pre installed layouts it is recommended to store the original layout before own modification are done and to save the modified file with a new name Additionally be aware to use the default file structure to save report files D Methods Report Layouts flexAnalysis If flexAnalysis is installed on a standalone system the path probably depends on the adjusted Regional Setting of the operating s
127. the corresponding menus shown in Figure 111 appear The content of the respective menu is a summery of specific commands and complies with the feasible handling of the window s content 2 8 2 Context Menus of the three flexAnalysis Windows Open Copy Copy i Cl A 2 Delete fm close 4 Auto Scaling Scale to Entire Spectrum Select Al Fia Clear All Unload Set Display Range Add to MS MS List E E rr Copy C Zoom MSMS List Ey Paste Ch Zoom Paste Special anu Set Selection Range Show always Identify Background Peaks Select All Bi Box Selection EX Layout View Analysis Set Color View Spectrum Reset Color Copy Calibration AL Find Mass List 42 Auto Scaling Replace Calibration dh Edit Scale to Entire Spectrum Th Edit Direct Set Color d e Clear Set Display Range Default Color Wi Smooth a zoom Properties ht Baseline Subtraction CA Zoom EX Edit Method Parameters Hide Y Axis Axis Font List Windows j Figure 111 Context menus of the Treeview Display and Peaklist window flexAnalysis 3 0 User Manual Version 3 0 2 109 Graphical User Interface GUI Bruker Daltonik GmbH All features except of Add to MS MS List Show Show always hide Y axis and Axis Font of the context menus are also available via buttons or one of the main menus Detailed information on the features is given in the respective section For information on Add to MS MS have a look in section 2 4 3 6 2 9 P
128. the user wants to add the Delete symbol xi To do so switch to the Command tab open the category Edit and transport the Delete symbol with the drag and drop to the new palette Customize CDT Toolbars Commands Categories i Buttons TE a Ba Analyse Method Annotation Select a category then click a button to see its description Drag the button to any toolbar Description Open multiple analyses with the Spectrum Browser Figure 98 The Commands tab The operator must be aware that in contrast to new palettes created by him the defaults can only be removed from the GUI but not deleted out of the program On choosing a user created palette the Reset button toggles to Delete 2 96 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI 2 4 10 10 Options The Options command and the corresponding icon of the Toolbar are used to manipulate optional settings for flexAnalysis The related dialog Figure 99 consists of four tabs Display Labels Export and Calibration Options aha Display Labels Export Calibration Drawing Optimization ott Local Max Stacked View Angle 45 Offset 3 s mm i ia l Windows 3 Figure 99 Option to specify the spectrum display 2 4 10 10 1 Display The group box Stacked contains the parameter Angle to arrange spectra in a specific angular position in the display whereas th
129. thod belongs to General information about Sax Basic Scripting can be found in the corresponding Help file For detailed information about Sax Basic Scripting please refer to the flexAnalysis Scripting Manual PN 227119 On launching the Script Editor flexAnalysis loads the script of the processing method currently assigned to the selected analysis The Script Editor s title bar shows the name of the open processing method and the analysis name in parentheses In case of a modified but not yet saved method the modified indicator is appended to the method name flexAnalysis 3 0 User Manual Version 3 0 2 67 Graphical User Interface GUI Bruker Daltonik GmbH Q analysis famethod flexcontrol generated xmass data c 2000 bruker daltai Wa x Script Edit View Debug Help BAS na Oe gt 25 EW Object General y Proc Main y Sub Main e Dim Spec Declare a spectrum object Set Spec Process nalysis Get the result spectrum of the processing Spec Enabled True Display result spectrum Save Save the analysis End Sub Function Process nalysis Dim Spec Declare a spectrum object Set Spec Spectra 1 Get the first spectrum of the analysis list Spec MassList RemoveAll Make sure that the peaklist is empty Set Spec Spec Process FindPeaks 800 4000 Find peaks between 800 and 4000 Da Set Process nalysis Spec return the result spectrum End Function Figure 77 Example of a Ba
130. to enter fixed atom ratios that should not be resolved For example when entering INS3P306 CHF for Min and IN3P306 C100H21F80 for Max then calculation will use exactly 3 N 3 P and 6 O but 1 to 100 C 1 to 21 H and 1 to 80 F e Chemical notation can be used For example CH3 6 will be resolved to C6H18 Allowed elements and minimum maximum atom numbers Lists the constraints to generate molecular formulas as specified in Min and Max i e the elements and minimum maximum atoms numbers allowed Within these bounds molecular formula calculation will occur Measured m z Enter the measured m z value for which possible molecular formulas should be calculated You can enter this value by typing it in or by selecting it in the currently active mass spectrum Position the Data pointer at the desired data point and click the left mouse button This enters the corresponding m z value Tolerance ppm Enter the error tolerance for the mass range to be analyzed in ppm This is a range within that flexAnalysis calculates the m z value of the molecular formulas The default value depends on the kind of the selected analysis For FTMS analyses default is 2 for ESI TOF analyses 4 and for all the other analyses 50 Charge For multiply charged ions enter the assumed charge the ion of the Measured m z carries Default is 1 Please consider the sign of the charge The mass of the electron is not negligi
131. u s New command to set up a new layout Note For detailed information to work with the ReportDesigner refer to the manuals on the installation CD File Edit View flexAnalysis Window Help E AR aea RADO ZE JALAO JAR i eps ae Tera e LLT SSE a Report _ oe TATA Ae For Help press F1 Figure 89 ReportDesigner with loaded report layout Creating a new layout by modifying an existing layout e Click rel or from the Tools Menu select ReportDesigner 2 80 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI e From the ReportDesigner s File menu select Open e In the Open dialog navigate to the layout file layout you want to use as a template to create a new layout and click Open This loads the selected layout e Modify the layout as desired e From the ReportDesigner s File Menu select Save As and save the modified layout to a new file name e If you don t want to use the ReportDesigner any longer select Exit from the ReportDesigner s File Menu Creating a new layout from scratch e Click or from the Tools Menu select ReportDesigner e From the ReportDesigner s File Menu select New e Setup the new report as desired e From the ReportDesigner s File Menu select Save As and save the modified layout to a new file name e If you do not want to use the ReportDesigner any longer select Exit from the ReportDesigner s Fil
132. uker Daltonik GmbH 2 4 10 11 Colors The Colors command is used to assign colors to particular screen elements in the Mass Spectrum Window Several Screen Elements are offered Newly added in this flexAnalysis version are Background Peak Label and Annotation label If peaks are recognized as backgrounds Identify Backgrounds Macro but not yet deleted from the peaklist they will be shown colored with the color selected here The annotations added via the new Annotation tool are also displayed in the color selected in this dialog Colors Screen Element EE eee eee cht tat io Custom colors a 160 Red jo if nn Jo Green 0 x E WE a Colors olid Lum 0 Blue 0 Figure 103 Colors dialog box 2 4 10 12 Glycopeptides For detailed information please have a look in the tutorial Quickstart_GlycopeptidesFA3 0 paf 2 4 10 13 Send Spectra to ProteinScape The Send Spectra to ProteinScape macro is used to send data to your ProteinScape server Applying this command opens the Spectrum Browser to select and load data 2 100 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI 2 4 11 Window Menu Window Compass Help Pg Analysis List Alt 1 ll Mass Spectrum Alt 2 Ed Mass List Alt 3 Sequence List Alt 4 Figure 104 Contents of the Window Menu 2 4 11 1 Overview Table 11 Features of the Window Menu Feature Window Menu Shortcut Description button
133. um Properties e Spot H 016 E 6 9 Chip 1 E warpz_ verify E we Chip O gt v Spot G 007 4 Spot G 008 e Spot H 007 e Spot H 008 6 9 Chip 1 Biotools_Flex_ISD_T3 Selected spectra 10 Open spectra 14 552 Cancel Select All Clear Selection Load all selected spectra Figure 12 Spectrum Browser Browse Select the folder from the hard drive with the data of interest Root Shows the selected path and folder e Filter Spectra Select checkboxes to filter the data type you want to open A tree structure on the left side of Figure 12 appears if the data structures correspond to the flexControl saving convention If the saving convention is not applied select the checkbox Unknown to let the data appear e From To Time filter to search for spectra e Spectrum Properties Shows the properties of a selected spectrum 2 6 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI e Spectra selected Displays the number of selected spectra Applying the button Clear Selection sets it to 0 e Spectra Shows the number of selected spectra in comparison to the total number of spectra found in the chosen root directory e Open Loads all selected spectra in flexAnalysis and closes the Spectrum Browser e Cancel Closes the Spectrum Browser without loading a file e Select All Selects all folders and subfolders of the chosen root directory e Clear S
134. und in the spectrum become colored in the spectrum display according to the chosen Background Peak Label color section 2 4 10 11 This filter feature might be useful for further processing e g database searches in BioTools xl 1563 7 1397 true 1588 7 1600 7 1603 7 1604 7 Figure 35 Assignment of background peaks true in the column Bk Peak If during an automatic run the background peak detection has been activated in the AutoXecute method the information which MassControlList is used is transferred to flexAnalysis The name of the Background list is shown in the spectrum Properties dialog Figure 36 appendtestW_C1112 Analysis Properties General Mass Spectrum Calibration Group Processing e Parameter Explanation Value Appli CL Mass control list used for background filtering Background 10 BaslsegHho Number of segments for baseline subtraction BaslSub Baseline subtraction applied lt lt o false al MEL Mass control list defined in AxsMethodE ditor for backoround filtering Background moot o Number of applied moothings T Figure 36 Background list information in the spectrum Properties 2 28 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH Graphical User Interface GUI 2 4 3 9 Remove Background Peaks The macro Remove Background Peaks is used to remove previously identified background peaks from the mass list and out of the display of the currently active spectrum To save the n
135. ves the previously identified background Background peaks from the peaklist Peaks Distances in Mass List s Macro to compare spectra 2 4 3 2 Find The Find command the corresponding icon A of the Toolbar and the short cut F5 are used to automatically generate a peak list for one or more selected spectra simultaneously Peak picking is performed according to the parameter settings of the current method section 2 4 7 6 1 The result is stored in the peak list and the mass spectrum is displayed with the detected mass peak labels flexAnalysis 3 0 User Manual Version 3 0 2 23 Graphical User Interface GUI Bruker Daltonik GmbH Only one peak list can be displayed at a time However more than one list can be re calculated at once by selecting the corresponding spectra with the mouse pointer in the Analysis List Window and simultaneously pressed lt Ctrl gt key of the key board Then activate the Find command for a peak finding in all the selected spectra 2 4 3 3 Edit It might become necessary to edit the peak list manually because too many or too few peaks were detected during the automatic peak find procedure The Edit command and the corresponding icon A of the Toolbar are used for interactively adding or removing peaks according to the respective peak detection algorithm AL A This command displays the Edit Mass List pointers iN or AA if the mouse pointer is A positioned in the Mass Spectrum Window The Add
136. ystem Otherwise the saved files cannot be located 2 4 1 14 Print The Print Command the corresponding icon of the Toolbar and the short cut Ctrl P open the print dialog Figure 20 A printout can be done from here with the button ok flexAnalysis 3 0 User Manual Version 3 0 2 13 Graphical User Interface GUI Bruker Daltonik GmbH Printer Mame HP Lazerdet 6P 6kmP PostScript Status Read Type HP Laseret 6P BMmP PostScript Wher pevhb eptd Comment Direct printing recommended for PostScript and PDF drivers 2 Use Printer resolution C Print with 300 DFI Print range Copies al Number of copies 1 Jy Report layout Layout MS Spectrum portrait Ww ReporDesigner Orientation 2 Portrait Landscape Figure 20 Print dialog 2 4 1 15 Properties The Properties command the corresponding icon of the Toolbar and the short cut Alt Enter open the dialog Properties that consists of the General page the Mass Spectrum page and the Calibration page All three tabs are displayed if the operator has previously selected only one spectrum in the Analysis List Window In case of multiple spectra selection only the General tab appears 2 14 flexAnalysis 3 0 User Manual Version 3 0 Bruker Daltonik GmbH pepmnd0_A 2443 Analysis Properties eee Title File name Date Instrument User name Method Sample Comment 1 Comment 2 Comment 3

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