NAIP workshopMANUAL copy
Contents
1. m z In the first case two ions are compared which are formed at the positions A and B in the preparation The electrical field that is seen by the ions decreases from the target to the extraction lens A Ion that is formed at an position above the target surface experiences a smaller field than in ion formed directly at the surface A shift of the apparent mass in the mass spectrum is observed between the two ions If the matrix preparation gets more inhomogeneous as shown in the second picture these mass shift increase the resolution decreases and the assignment of the true mass becomes more difficult and requires to sum up a lot more spectra to compensate for that statistical error Selection of the Matrix For proteins and peptides the most commonly used matrices are 4 hydroxycinnamic acid a Cyano HCCA CCA Sinapinic acid and Dihydroxy benzoic acid DHB All Matrices have different pros and cons The matrix substances should be of highest purity Please note that the matrix substances that are obtainable from common suppliers are usually not pure enough to give the best possible result in MALDI MS HCCA should be pale yellow crystals Sinapinic acid should be almost white and DHB white crystals Matrix substances especially purified for MALDI are obtainable from BRUKER 11 a Cyano 4 Hydroxycinnamic acid This Matrix is commonly used for peptides in the lower mass range2. Copright Howy to Obtain Su Ort Contents i File Edit MassList Process Calibrate Annotation Method FAST wiew Tools Window Compass Help LAMAR dh Q 2 Q Q iw B 2 Mass Spectrum Intens jau E cytochrome cAD 18 T5Ref Smoothed Baseline sub 9000 ACTH LP PEPMDAD 15Lin ACTH 1 39 LP 210 6000 4000 2000 ko 4 Dwed Seq x H 5283 3 728 63 6313 2 237 33 5388 1 2 312 For Help press F1 A he o ud 5173 5 BDDD 8000 cytochrome _0 B18 1 21 ES LL i 10000 122585 Eylnchrame ci SLin Smoothed Baseline sut 120138 129737 12000 Range Selection 14000 RAJESH 16000 18000 y 27 7 Bruker Daltonik GmbH 6 Sequence Database Searches from MALDI Peptide Mass Fingerprints PMFs 6 1 eaten cts des 6 1 6 2 Sequence Database Search using a MALDI Peptide Mass Fingerprint 6 2 Gis _ Seiinelic 6 3 6954 eet ni RE VOR i 6 6 6 3 2 Introduction to MASCOT 2 0 Query Results Interface 6 7 6 3 3 Work with Search Results in BioTools 6 11 6 1 Introduction The most frequent task in pr
3. Mase Tol M5 ppm Mass values MH Monoisotopic Average Data file Query Data gar 433970 1078 071700 1163 600850 477 008840 1283 694050 441 938320 1362 656610 352 614460 1439 808870 3309 051950 Search unmatched peaks only Instrument MALDI TOF TOF Results Overview Report top 20 bits check matching MSM only Copy Peaklist Copy Masslist Save as default Exit Figure 6 2 5 search dialog for the internet search Tutorials for BioTools Version 3 2 6 3 Bruker Daltonik GmbH Sequence Database Searches from MALDI Peptide Mass Fingerprints PMFs Specify the information as in Figure 6 2 Push the Start button fora MASCOT search Tutorial library searching and the BioTools search interface Typically Fixed modifications include the known chemistry such as reductive disulfide cleavage and carbamidomethylation i e reaction with iodoacetamide Unknown chemistry such as the artifact Methionine oxidation or phosphorylation can be specified as variable modifications Allowing for optional modifications may reduce search specificity but some like Protein N term Acetylation Formylation Pyroglutamylation may help identification of small proteins Also labeling chemistry such as ICPL light and ICPL heavy or ICAT light heavy are specified as variable modifications for quantitative proteomics experiments Mass tolerance MS the peptide mass error is important a
4. MH mono 2061 828 MH avg 2062 968 MS MS Tol 0 500 Da v Peaks 89 Above Threshold 89 Zoom to m z for 2 Masses Monoisotopic Average Calculate Masses v Threshold 0000 Assigned 26 Not Assigned B3 1 m z 0 00 lonji 2 3 4 5 6 7 8 9 1011 12 13 14 15 16 1 120 081 248138 335171 464214 593257 F es E E ae a m E D E L G D K 721315 849374 977432 1158446 1287489 14 FOS EE QQ ED EL Q DK 148076 276134 363168 492209 B21251 749310 8773869 1005427 1186441 1315484 14 F e s E E a a a T E D E L G D K 147 113 262440 390198 503282 632325 747352 876395 1057 409 Eu 1313 526 14 F e s E E a o a E D E L IG D K 120 081 101071 60044 102055 102055 101071 101071 101071 154 026 402055 16154443424140 9 8 7 8 5 4 3 2 1 Lys Asp Gn teu Gu Gu Tr Gn Gn gt 3 INSTRUMENT LAYOUT Bruker flex systems such as the Ill are always composed of only two main components 1 Mass spectrometer twister optional 2 Data system chapter 3 2 3 1 Schematic of the Mass Spectrometer Figure 3 1 shows the principle the ultraflex Ill mass spectrometer works 1571 152 Ground potential 151 Linear detector Target Reflector Reflector plate detector Figure 3 1 Schematic of the instrument Sample Preparation for MALDI TOF Samples for MALDI TOF analysis need to meet certain requirements for obtaining good spectra
5. General remarks The chemicals should be of highest available purity Saturated matrix solutions should be prepared freshly by sonication It is important to spin down the remaining undissolved matrix in a centrifuge The supernatant should be aspirated carefully to avoid aspiration of crystals Dried droplet method Typically a saturated matrix solution is prepared Unless otherwise noted the solvent used 1s TA 33 Acetonitrile 0 1 This matrix solution is mixed in equal volumes with the sample solution The sample solution should be acidic since basic conditions will dissolve the matrix The mixture is pipeted on the target 0 5 to 1 ul and dried at ambient temperature The preparation will yield relatively large crystals on the target surface a well as regions without matrix or analyte The advantages of the dried droplet method are The method 15 suitable if the sample contains organic solvents If a sweet spot is found on the preparation a large number of laser shots can be applied to that spot If the sample contains contaminants there is a chance that analyte and contaminants will crystallize at spatially different regions on the target The sample can be washed after the crystallization to remove salts The sample can also be recrystallized after washing Disadvantages include the need to search for sweet spots and the limited resolution due to the large crystals Thin layer methods This method 15 applicab
6. INTERNATIONAL CENTRE FOR GENETIC ENGINEERING AND BIOTECHNOLOGY NEW DELHI COMPONENT INDIA National Agricultural Innovation Project NAIP supported workshop on Proteomics Use of Mass Spectrometry in Biology 23 4 27 March 2009 Graphical User Interface GUI A an Eral TOFITOF _25 par Be DHDA Mew Ib Hip m Menu Bar Title Bar ewn amp Mb 510 F Button Bar cu 2 E Eti Lan f 5 rn Ff 100 eh pate ir F E ue 1n 1500 i Er sho T0 JOD 2 3 Suka El Hone 5 hol fienda anum Comer Dacin Paca Tec Sek Calton uan Method Dat hsnas H Bample Carrier I amp Cee Prepase Fa grows S pecinan T DEPT 10 Panne SEX Fer praua F 10 PROTEIN SAMPLE PREPARATION USING 2 D CLEAN UP KIT Source G E Healthcare Transfer the protein samples into tubes that can be centrifuged at 8000 _ g Each tube must have a capacity at least 12 fold greater than the volume of the sample Use only polypropylene polyallomer or glass tubes The wash buffer used later in the procedure is not compatible with many plastics This limits the choice of centrifuge tube materials For each
7. Protein View Match Protein 83 Flex BSA dig For Help press F1 Figure 6 10 Imported Search Result in BioTools tree view The most important information in the tree view is the list of peptides in the particular sequence which match the experimental masses at the specified conditions of the search Each peak entry may consist of measured m z calculated intensity deviation mass error Da ppm sequence range of the peptide partials number of missed internal cleavage sites and the sequence The actual parameters that are displayed can be customized as described in the BioTools User Manual in chapter Useful Hints Treeview Window Context Menu Selection of the tree view entries at any level can be visualized in the spectrum as well as in the sequence view underneath the spectrum Either single peaks or a set of peptides matching a protein Digest matches can be selected black numbers with additional sequence position information indicate matched peptides Peptides which contain an optional modification are color coded in blue Tutorials for BioTools Version 3 2 6 13 Bruker Daltonik GmbH Sequence Database Searches from MALDI Peptide Mass Fingerprints PMFs The Sequence Viewer Simultaneous to the treeview information the sequence is also loaded into the Biolools Sequence Viewer following the Get Result s operation in the MASCOT re
8. DFAEDEDVCK EECCAEDDPH NRLCVLHEET TFHADICTLP CCAADDEEAC EHFKGLVLIA HTLFGDELCE FDFNTLCDEF CCOAEDEGAC RLSQKFPEAE ODTISSELEE NYOEAEDAFL ACYSTVFDEL VSTPTLVEVS PYSEEVTECC PTEEOIEK0T FAVEGPELVV 4 F E Fertig fas Lokales Intranet 2 Protein view to visualize sequence coverage of matching peptides Tutorials for BioTools Version 3 2 Bruker Daltonik GmbH Define the Search Mascot Search Results Protein View Microsoft Internet Explorer Datei Bearbeiten Ansicht Favoriten Extras zurdck mb fx fat EA Suchen sg Favoriten Medien y Adresse wiw matrixscience comicoiprotein_ view pl Til amp dabkaf20040913 FaErmbY e dab amp hik 1 229 235 820 49 819 48 819 46 0 02 236 245 1145 63 1144 62 1144 64 0 01 AWSVARLSU UE 402 412 1305 69 1304 69 1304 71 0 02 HLVDEPOHLIK 421 437 2028 09 2027 09 2027 10 0 01 LGEYGFUHALIVRYTRE 438 455 1897 07 1896 07 1896 07 0 00 VPQVSTPTLVEVSRELGE 549 557 1014 61 1013 60 1013 61 0 01 QTALVELLE 569 580 1399 70 1398 69 1398 69 0 00 TWMEHFVAFWDE match to 924 50 1017 54 1142 66 1547 52 1917 06 2435 15 2553 12 2554 12 2555 14 2 Error Cppm 1 aa gon 1200 1500 2000 2400 2000 RMS error 17 ppm Mass gt P1 AdAN 1752 4 Bos taurus serum albumin alb mRMN amp R complete cds Bos
9. The more careful you prepare samples including early steps of isolation and preparation the more likely a successful analysis will be Here are some guidelines of which kind of treatment 15 advantageous for mass spectrometric analysis and which is not Avoid the use of non volatile agents like salts NaCl CaCl2 KH2PO4 detergents Tween Triton SDS chaotropic agents Urea Guanidinium salts and non volatile solvents like DMSO DMF or Glycerol If you can t avoid these agents purify Dialysis ZipTips and RP HPLC are good purification methods if you use volatile solvents and buffers e g 0 1 v v trifluoracetic acid 10 mM After purification lyophilize if possible Ion exchange beads may work well for salt removal Suitable solvents are ones that are volatile For sample work up and purification water ammonium hydrocarbonate ammonium acetate ammonium formiate acetonitrile trifluoroacetic acid Quantitate the sample you are going to provide for analysis by methods like photometry e g OD Bradford assay and ELISA HPLC 1s useful since it allows for purification and quantitation in a single procedure The range for many samples preparations 15 not very large therefore it 1s necessary to have a good estimate of the sample amount because the sample amount may need to be varied on the target The total amount of sample needed for MALDI analysis depends on the sample type For small mw peptides 1 000 or les
10. 100 mM buffer and 100 Acetonitrile ACN solution IV _ They were briefly rinsed with 100 ACN solution and were dehydrated with 100 ACN for 20 mins at RT with gentle agitation The solution was then discarded and the gel pieces were air dried or dried in a SpeedVac 3 Reduction and Alkylation The following step is required for 1D SDS PAGE gel piece and not for 2D SDS PAGE i The gel pieces were incubated with Dithiotrietol DTT 10 mM DTT prepared 11100 mM NH4HCO 3 solution at 56 C for 45 mins for reduction The sample was then cooled to room temperature and the DTT solution was removed sample was then alkylated by treating with Iodoacetamide IAA 50 mM IAA in 100 mM solution in the dark at RT for 30 mins ii They were washed with 100 of 1 1 100 mM NH4HCO z buffer and 100 Acetonitrile ACN solution for 15 mins iv They were then rinsed briefly with 100ul of 100 Acetonitrile and were dehydrated for 20 mins at RT with 100 ACN and the solution was discarded and the gel pieces were air dried or dried in a Speed Vac 4 In gel Digestion Trypsin or Chymotrypsin enzyme solution prepared in 50 mM NH4HCO3 was added to the gel pieces and they were rehydrated for 60 mins at 4 C and following rehydration 30 50 of 25 mM buffer was added 1f required and incubated for 16 to 18 hrs at 37 C NOTE The gel pieces should completely remain immersed in the buffer during incubatio
11. 673 267 280 2492 261 45 65 Protein 02769 Serum albumin precursor Allergen Bos d 5 BSA ALBU_BOVIN Peak threshold 0 0 Intensity coverage 96 4 44452 cnts Sequence coverage MS 25 7 Sequence coverage MS MS MEWVTFISLL 100 HTLFGDELCK 190 ANKYNGVF QE 280 GDLLECADDR 370 HPEYAVSVLL 3 LLFSSAYSRG 110 VASLRETYGD 200 CCOAEDEGAC 290 ADLAKYICDN 380 RLAKEYEATL 460 RSLGKVGTRC ga 550 DTEKOIKKOT 470 CTKPESERMP 560 VFRRDTHKSE 120 MADCCEKQEP 210 LLPKIETMRE 300 QDTISSKLKE 390 EECCAKDDPH 480 CTEDYLSLIL 570 IAHRFKDLGE 130 ERNECFLSHK 220 KVLASSAROR 310 CCDKPLLEKS 400 ACYSTVFDKL 490 NRLCVLHEKT 580 ALVELLKHKP MENFVAFVDK 0 0 pl 5 8 MW kDa 63 90 EHFEGLVLIA FSOYLOOCPF DEHVELVNEL TEFAKTCVAD ESHAGCEESL nh 140 DDSPDLPELK 230 LRCASIQKFG 320 HCIAEVEEDA 410 KHLVDEPONL 500 590 CCAADDEEAC 150 PDPNTLCDEF 240 ERALKAWSVA 330 IPENLPPLTA 420 IKONCDOFEK 510 TESLVNRRPC 160 KADEKKFWGK 250 RLSQKFPKAE 340 DFAEDKDVCK 430 LGEYGFONAL FSALTPDETY 170 180 YLYEIARRHP YFYAPELLYY SSS 270 FVEVTELVTD LTEVHKECCH 360 NYQEAKDAFL GSFLYEYSRR 440 450 IVRYTRKVPO VSTPTLVEVS 540 WPEAFDEKLF TFHADICTLP 600 FAVEGPELVV 610 STOTALA For Help press F1 Figure 6 11 NUM suckau Imported
12. These adducts can be resolved in the mass spectrum for proteins up to 40 kD DHB This 15 the Matrix of choice for the preparation of glycoproteins and glycans It is also often times used for Peptides Unlike a Cyano and Sinapinic Acid it is soluble in water as well as organic solvents The main disadvantage of DHB ist the fact that it forms big crystal needles This means that the geometry of the sample changes from spot to spot on a preparation If spectra are summed up from different spots on the sample preparation the resolution 15 considerably lower than spectra obtained from an preparation On a steel target DHB preparations will form a crystalline ring Good peptide spectra are usually only obtainable from the rim of that preparation The main advantage of DHB for MALDI of peptides is the fact that this matrix is more tolerant towards contaminations such as salts and or detergents than other matrices Typical preparation of DHB A rim of large crystals is formed best peptide spectra are usually obtained at the rim 12 Preparing the sample on the target Like the choice of the matrix substance there is also a choice of how to actually prepare the sample The advantages and disadvantages are discussed in the following section The methods discussed here apply for conventional targets Anchor targets have to be prepared using specialized anchor chip protocols Please refer to the anchor chip manual for those protocols
13. This matrix is not soluble in water and well soluble in organic solvents It 1 considered a hard matrix which means the analyte molecules get a lot of internal energy during desorption and ionisation This leads to a considerable amount of 10n fragmentation in the drift tube post source decay If peptides of small molecular weight are measured and the laser power is chosen only slightly above the threshold this is not a problem If the analyte molecules become bigger however the probability of the fragmentation increases until almost all of the analyte ions undergo fragmentation Therefore a Cyano is the matrix of choice for PSD analysis The main advantage of a Cyano the measurement of peptides 15 the ability of this matrix to form small homogenous crystals Since geometric inhomogeniety relates directly to decreased resolution in the MALDI analysis a Cyano preparations usually yield good resolution Since HCCA 15 insoluble in Water the samples can be washed on the target Sinapinic Acid Sinapinic Acid is most commonly used in the analysis of high mass proteins It is also not soluble in water but well soluble in organic solvents Compared to a Cyano it is a softer matrix The analyte Ions get less internal energy and the amount of fragmentation is smaller making this matrix more suitable for measurement of proteins Sinapinic Acid also can form small crystals However Sinapinic Acid tends to form adducts with the analyte ions
14. http matrixscience com cgi protein_view pl fil amp idata 20040913 FaErmby erat TRIX Mascot Search Results CIENCE Protem View Match to AF547068 HID Mominal mass AAH178724 Score 190 Expect 3 Bos taurus Mj 71274 Calculated pl value 3 L5 NCBI BLAST search of 2441178524 against nr Unformatted sequence string for pasting into other applications Taxonomy Bos taurus Links to retrieve other entries containing this sequence from NCBI Entrez CAA41735 from Bos taurus CAA76537 from Bos taurus Fixed modifications vVariahle modifications Cleavage by Lys c Sequence Coverage Carbamidomethyl Oxidation M c 82 cuts C term side of E unless next residue is HMumhber of mass values searched 22 Wumber of mass values matched 13 235 Matched peptides shown in Bold Red 1 51 101 151 201 251 301 351 401 451 901 351 601 Figure 6 7 6 10 MEWVTFISLL FSOYLQQCPF VASLRETYGD EADEKEFWGE LLPEIETMRE FVEVTELVTD CCDEPLLEES GSFLYEYSRR EHLVDEP HI RSLGKVGTRC TESLVNRRPC ALVELLKHEP STOTALA LLFSSAYSRG DEHVELVHEL MADCCEREGQEP YLYEIARRHP EWLTBGAROR L TEUWHRECCH HCIAEWEEDA HPEYAVSVLL IKONCDOFEEK CIKPESERHP FSALTPDETY EATEEOLERTW WFRRDTHESE TEFARTCV AD ERNECFLSHE FYAPELLYY LECASIGQEFG GDLLECADDE IPENLPPLTA RLAEEYEATL LizEY GF QHAI CTEDYLSLIL WPEAFDEELF MEHFVAFVDE IAHRFEDLGE ESHAGCEESL DDSPDLPELEK AHEYNGVFOE ERALEAWSVA ADLAEYICDN
15. mass axis are provided additionally This interface enables you to interactively judge the data from a mass spec point of view mass errors signal shape intensity isotopic distribution and from a protein chemical view Distribution of cleavage sites in the protein hot spots in the sequence indicated by several peptides sharing the same cleavage site etc It is basically a result editing board from which you can initiate various further investigations and to which the respective results are reported to for your further judgment Tutorials for BioTools Version 3 2 6 15 Bruker Daltonik GmbH Sequence Database Searches from MALDI Peptide Mass Fingerprints PMFs D BioTools D Data Biotools Tutorial data Flex BSA_digest 0_M8 1 1SRef pdata 1 1r Oi x File Edit Yiew Analysis Search Tools Window Compass Help 18 MS MS ls so Q 1 URN GO 1888 924 1479 792 1567 742 169 183 421 433 347 359 V ly Digest Matches Seow 1880 927 1439 809 508 523 360 371 1724 840 2045 028 1362 657 469 482 168 183 89 100 1667 809 1823 897 1283 694 469 482 508 523 361 371 peak 10 nM peak 11 1639 943 1749 673 1163 601 437 451 2492 261 66 75 45 65 peak 13 j peak 14 oM peak 15 738 BTDXom BTDX xml 8 0 MEXINIS EE p a
16. t 17 2492 264 1 354 1000 1250 1500 1750 2000 2250 miz Protein View Errors MSMS fragments MSMS Analysis Protein 02769 Serum albumin precursor Allergen Bos d BSA ALBU_BOVIN Peak threshold 0 0 Intensity coverage 96 4 2 44452 cnts Sequence coverage MS 25 7 Sequence coverage MS MS 0 0 58 MW 69 10 20 30 40 50 60 70 60 90 MKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFEDLGE EHFKGLYLIA FSOYLOOCPF DEHVELVNEL TEFAETCVAD ESHAGCEKSL 100 110 120 130 140 150 160 170 180 HTLFGDELCK VASLRETYGD MADCCEKQEP ERNECFLSHK DDSPDLPELK PDPNTLCDEF KADEKKFWGK YLYEIARRHP YFYAPELLYY l p 190 200 210 220 230 240 250 260 270 ANEYNGVFOE CCOAEDEGAC LLPKIETMRE EVLASSAROR LRCASIOEFG ERALKAWSVA RLSOKFPKAE FVEVTELVTD LTEVHEECCH 280 290 300 310 320 330 340 350 360 GDLLECADDR ADLAEYICDN ODTISSELEE CCDEPLLEES HCIAEVEKDA IPENLPPLTA DFAEDEDVCK NYQEAKDAFL GSFLYEYSRR p du 380 390 400 410 420 430 440 450 HPEYAVSVLL RLAEEYEATL EECCAEDDPH ACYSTVFDKL KHLVDEPONL IKONCDOFEK LGEYGFONAL IVRYTREVPO VSTPTLVEVS M gS OE 360 470 480 490 500 510 520 530 540 RSLGKVGTRC CTKPESERMP CTEDYLSLIL NRLCVLHEKT TESLVNRRPC FSALTPDETY VPKAFDEKLF TFHADICTLP a E E 550 560 570 580 590 600 610 DTEKQIKKOT ALVELLEHEP MENFVAFVDE CCAADDKEAC FAVEGPELVV STOTALA mn 4 0 M S For Help pre
17. 02 34312 HLVDEPONLIE 1399 70 1395 69 1398 69 0 00 558 TVMEHFVAFVDE 1897 07 18596 07 1596 07 0 00 338 455 VPOVSTPTLYEVSRSLGE 2028 09 2027 09 2027 10 0 01 Tal 357 LGEYGFONALIVEYTRE 2953 39 2952 36 2952 50 D 12 161 183 Y LYETARRHPYFYAPELLYYANE EE ym Ho match to 924 50 1017 54 1142 66 1347 52 1917 06 2435 16 2553 12 2554 12 Figure 6 6 Protein Summary Report index and matching peptides Under Results List for each entry there is a detailed list of all matching peptides and the actual mass error in Da Delta irrespective of error dimension in the search Through the hyperlinked access number you open the Mascot Protein View which contains a peptide coverage map of the full protein and all available information about the database entry Figure 6 7 Also an error plot is provided which allows a simple mass error evaluation Figure 6 8 A gross mass accuracy value provided here as well as in BioTools 3 2 see Figure 6 12 is the RMS error that is well suited to give the average mass error of that dataset Tutorials for BioTools Version 3 2 6 9 Mascot Search Results Protein View Microsoft Internet Explorer Datei Bearbeiten Ansicht Favoriten Extras 7 Bruker Daltonik GmbH Sequence Database Searches from MALDI Peptide Mass Fingerprints PMFs zur ck p fx fat suchen 3 Favoriten EP Medien cs Eh cb EW e Wechseln zu Adresse 2
18. 1 ir The red histogram like peaks indicate the mass intensity values from the mass labeled spectrum if View Picked Peaks is selected Se BioTools D Data Biotools Tutorial data Flex BSA_digest 0_M8 1 1SRef pdata 1 1r l x js File Edit View Analysis Search Tools Window Compass Help 8 z ws so Tree hierarchy Calc M Dev p Abs Int 1000 D Data Biotools T utorial d E M 4 Unmatched El Peaks f tha M peki 1888 924 peak 1479 792 1567742 169 183 J M G P02769 Serum album 421 433 347 359 i E Digest Matches 1880 927 Search Parameter Ch peak 2 1163631 25 587 1439 809 508 523 j peak3 1283 711 12 890 360 371 peak4 1362 672 11 421 1724 840 peak5 1439 812 1 959 3 469 482 1473795 2 317 1362 657 peak 8 1567 743 0 688 89 100 j peak 9 1533938 3282 i 1667 809 1823 897 peak 10 1667 813 2 633 469 482 peak 11 1724 835 2 981 i 1283 694 508 523 cM peak 12 1749 662 6 120 361 371 peak 13 1823 900 1 676 1639 943 1749 673 14 1880 921 3 373 i 1163 601 437 451 267 280 2492 261 V j 15 1888 927 1 335 66 75 T 45 65 16 2045 028 0 066
19. 15 Queries matched 13 AF542068 NID Bos taurus Query ABBOS Mass 71221 Score 169 Expect 42 13 Queries matched 12 Stop serum albumin precursor validated bovine ABSHS Mass 71139 Score 52 Expect 0 22 Queries matched 5 New serum albumin precursor sheep Exit 240802 Mass 72087 Score 33 Expect 17 Queries matched 3 protein tyrosine kinase 2 7 1 112 syk 72K chain pig LiKG4954 Mass 103355 Score 27 Expect 59 Queries matched 3 AY037947 NID Oryctolagus cuniculus S13269 Mass 45490 Score 26 Expect 73 Queries matched 2 translation initiation factor eIF 4A I rabbit fragment AAB38527 Mass 17863 Score 24 Expect 1 32 02 Queries matched 2 CHU3S 4274 NID Capra hircus iNSA Mass 45327 Score 23 Expect 1 5 02 Queries matched 2 procarboxypeptidase h pig CBPB PIG Mass 46141 Score 23 Expect 1 6 02 Queries matched 2 Carboxypeptidase precursor 3 4 17 2 Sus scrofa Pig of h Figure 6 4 Concise Protein Summary Report If you format the report as Protein Summary you may select to add a peptide match overview Figure 6 5 which allows to check the identity of matching peptides across the candidate sequences Red circles indicate identical sequences if one of them is under the mouse cursor This is very useful to get a feeling for the relationship among the retrieved sequences Tutorials for BioTools Version 3 2 6 7 Bruker Daltonik GmbH Sequence Database Searches from MALDI Pept
20. Search Result in BioTools sequence viewer Important information about the global match between spectrum and sequence is displayed in the header of the sequence viewer 6 14 Tutorials for BioTools Version 3 2 Bruker Daltonik GmbH Define the Search Protein shows the database entry information The values for isoelectric point pl and molecular weight MW kDa are based on the protein sequence solely no modifications etc are considered in these calculations The Intensity coverage provides an idea about the fraction of the intensity of all matched peaks vs the total picked peak intensities which are related to the selected protein A coverage of larger than 80 means that you achieved a fairly complete extraction of information from the spectrum while a coverage of 20 means you probably missed the point in analyzing the spectrum so far or contaminations are significant Sequence coverage MS is the fraction of the annotated sequence in a mass fingerprint vs the total sequence length In MALDI fingerprints this value typically varies between 10 and 90 depending on protein size and data quality good quality spectra of small proteins may yield 90 while larger proteins like BSA will yield only 15 30 In the Match Errors tab the error plot is shown Da or ppm scale can be selected which allows a simple mass error evaluation Figure 6 12 The average RMS error always ppm and the regression function of the errors along the
21. aa rry skt C Protein View Match Errors MSMS fragments Analysis RMSError 0 01 Da linear regression f X 0 032778 0 000017 X Correlation quotient 0 560067 0 02 03 1100 0 1200 0 1300 0 1400 0 1500 0 1600 0 1700 0 1800 0 1900 0 2000 0 2100 0 2200 0 2300 0 2400 0 Figure 6 12 Imported Search Result in BioTools match errors the y axis may be switched to ppm scale by right mouse button click on the y scale 6 16 Tutorials for BioTools Version 3 2 Bruker Daltonik GmbH Define the Search The basic observations assumptions to explaining them and the possible procedures to check them are Problem 1 Many peaks remain unaccounted for after import of a search result Intensity coverage poor Assumption 1 There are more proteins in the mixture and didn t find them all yet repeat the MASCOT search and select the Search unmatched peaks only option in the search dialog Assumption 2 Several peaks may match actually to the protein but not in the simple way assumed for database searching want to check for higher mass deviations tolerate more incomplete digestion or even unspecific cleavages typically trypsin gives raise to further peptides resulting from cleavage after Y W L and I You may even want to check for the presence of various suspected modifications or sequence errors or point mutations For further work on the identification of the unmatched peaks at this stage of analysis p
22. ability Based Mowse Score Tens score is 1U Log P where P is the probability that the observed match is a random event Protein scores greater than 58 are significant pc 05 n SS Number of Hits 5 7 Z go 120 150 200 Probability Based Mowse Score Figure 6 9 MASCOT search results Mowse score To clear the BioTools treeview from previously imported search results press the Clear button before you import the new data For further searches which do not include the peptides matching the first protein sequence press New to return to the Search dialogue window and check Search unmatched peaks only in the Mascot search dialog Figure 6 2 To exit the Query result page to continue working with the spectrum in BioTools press Exit 6 12 Tutorials for BioTools Version 3 2 Bruker Daltonik GmbH Define the Search The Treeview The treeview on the left side contains the data file information with up to two info lines these contain the comments 1 and 2 provided by the operator during spectra acquisition The unmatched peaks as well as the peaks matching the identified sequences are listed Information from the imported MASCOT search the list of sequence names which were retrieved with a sublevel called Digest matches which contains the MASCOT score This is followed by the basic information about the search parameters and the chemical modifications specified K biotools D data Bi
23. cation 6 4 Tutorials for BioTools Version 3 2 Bruker Daltonik GmbH Define the Search does If higher values seem to be required on a routine basis you need to optimize your digest for more complete proteolysis In silver gels destaining might help After you set up the search parameters for a type of application you need to process frequently push the Save as default button to store conditions Every spectrum is searched with this set of default conditions initially If you then modify the conditions for a particular spectrum the next time you open the spectrum these previous parameters will appear allowing you to reproduce the last accepted result Push Copy mass list to paste the mass list into the clipboard Note From the clipboard you can paste them into any browser based search engine on the web such as PeptideSearch PepSea Profound or MS Fit The search results from these programs however cannot be imported back into BioTools in contrast to MASCOT Push Copy Peaklist to paste the list of masses and intensities into the clipboard lf a search result is already imported into BioTools and a significant number of unaccounted peaks suggest the presence of another protein in the digest mixture check the Search unmatched peaks only option Now only those peptides of the tree view category unmatched are used for this 2 round of searching Note this approach simplifies the setup of a secondary search but may cause
24. cification If it fails Assumption 1 The peak is related to an interesting since unknown structural detail of my identified protein Search for those masses in protein sequence and allow all thinkable modifications to occur and even allow tolerating single position sequence variations Use the MS MS spectrum to judge the calculated suggestions please refer to the SequenceEditor Tutorial Protein Digests chapter P 2 Perform Enzymatic Digest chapter P 3 Format the Digest Results and chapter P 4 Export Digest Results to Spectrum Alternatively RapiDeNovo and MS BLAST may help either as a local BLAST search or via the internet Assumption 2 The peak is related to another protein which hasn t been identified in the mass fingerprint and it is not in the protein database Try searching the ESTdb at the matrix science homepage first and DeNovo sequencing second 6 18 Tutorials for BioTools Version 3 2 22
25. core Tens score is JU Log P where P is the probability that the observed match 15 a random event Protein scores greater than 28 are significant p 05 5 WH x X Humber of Hits TT gt S S N S S S S N 7222277 tye E 120 160 o0 Probability Based Mowse Score Figure 6 3 MASCOT search results overview To get a short introduction of the MASCOT Query results interface continue with section 6 3 2 Introduction to the MASCOT 2 0 Query Results 6 6 Tutorials for BioTools Version 3 2 Bruker Daltonik GmbH Define the Search 6 3 2 Introduction to the MASCOT 2 0 Query Results Interface Since there frequently is more than one homologue or near identical sequence splice variants etc even in nonredundant databases the search result may be obscured MASCOT offers for this case the Concise Protein Summary Report Here the sequences and scores of the highest scoring sequence for each cluster of homologue sequences are shown This is the preferred mode to view the results r Move Concise Protein Summary Report Back Format Concise Protein Summary Help Significance threshold p lt 0 05 Max number of hits 0 Re Search All Search Unmatched Get All Get Hit s 1 44451311 Mass 71244 Score 190 Expect 3 2 15 Queries matched 13 BOVALBUMIN NID Bos taurus 441417824 Mass 71274 Score 190 Expect 3 2
26. e nitrocellulose may yield interfering signals in the lower mass range of a peptide spectrum especially if a high laser power is used thin ie of H H CCA Double layer method One way to combine the advantages of dried droplet and thin layer preparation is the double layer method Here a thin layer of matrix is prepared as described above and on top of that thin layer a normal dried droplet The small crystals in the thin layer act as crystallisation nuclei for the dried droplet The result 1s a homogenous preparation that is well suited for automatic measurements The number of spectra that can be acquired from one specific spot is higher than in the thin layer method but not as high as in dried droplet The preparations can be washed but recrystallization would convert the preparation into a normal dried droplet preparation steel surface Grains wal dem bus t Jar uri inan EC Te ra eni Rear i 4 Em iy Md y B z bue yn et gt N HA ny ca WI cg ge a cia odi du a p oar NA acb rt Pa M psal double le layer of HC CA 14 Preparation Improvement Washing and Recrystallisation Washing the preparation During the preparation of the target 1t is possible that contaminants and sample crystallise at different positions spatial separation Especially salts have usually a higher solub
27. ery results gt Move Index Back Accession Score Description AAH17824 71273 190 AF542068 NID Bose taurus AAA51411 71233 190 EBOWALBUMIN NID Bos taurus F esulta 1 2 3 ABBOS 1221 169 serum albumin precursor validated bovine 4 ABSHS 71139 52 serum albumin precursor sheep Clear 5 ABPGS 71362 42 serum albumin precursor pig fragment Get Al AAA30988 71345 42 PIGALBA NID Sus scrofa 7 BA 02822 16504 34 T cell receptor delta chain Fragment Bos taurus Bovine Get Hits 8 4140802 72087 33 protein tyrosine kinase EC 2 7 1 112 syk chain pig 9 540161 28043 28 phosphoprotein phosphatase 3 1 3 16 3 alpha catalytic cha 10 C36222 arwyd phosphoprotein phosphatase 3 1 3 16 3 heta catalytic chair Results List 1 AAMi7824 Mass 71274 Score 190 Expect 3 2 15 Queries matched AFS42068 NID Bos taurus bserved Mriexpt Mr calrs Delta Start End Miss Peptide 620 49 519 45 819 46 0 02 229 235 FGERALE 205 9 205 45 905 46 0 02 205 211 IETMEEE gzz 45 921 47 921 46 1 205 211 Oxidation M 974 45 973 44 973 45 0 01 a7 44 DLGEEHFE 987 55 956 54 Sob 53 0 0i 29 36 SEIAHRFE 1014 61 1013 60 1013 61 0 01 549 e 557 1145 63 1144 62 1144 64 0 01 236 245 AWSVAELSQE 1163 60 1 60 1162 62 0 03 75 LWNELTEFAE 1305 69 1304 69 1304 71 0 02 4
28. esuspension Centrifuge the tubes at 8000 _ g for 10 min to remove any insoluble material and to reduce any foam The supernatant may be loaded directly onto first dimension transferred to another tube and stored at 80 C for later analysis TWO DIMENSIONAL GEL ELECTROPHORESIS Source G E Healthcare SAMPLE PREPARATION The Protein sample was incubated with Rehydration buffer at room temperature for four hours minimum A pinch of Bromophenol blue was added to the rehydrated protein sample and the sample was transferred to the rehydration tray The IPG linear or NL strips were then placed over the protein sample with the gel side facing downwards The set up was left undisturbed for 20 minutes and then mineral oil was poured over the strip The strip was left for rehydration for 12 16 hrs overnight at room temperature The excess oil in the strip was blotted and 1t was transferred on to a focusing tray manifold or strip holder The strip containing the protein was positioned with low pH at the ve side and high pH at the ve side and then mineral oil was added FIRST DIMENSIONAL SEPARATION For the first dimensional separation the IEF Iso Electric Focussing was performed according to the manufacturer s manual with slight modifications EQUILIBRATION OF THE SAMPLE Strips were then equilibrated in a buffer containing 50mM Tris HCl pH 8 8 7 urea 2M thiourea 20 glycerol 2 SDS 65mM DTT for 15 20 mins with c
29. for at least 1 at 20 C to each tube For initial sample volume of 0 1 0 3 ml add 1 ml of wash buffer However the volume of wash buffer must be at least 10 fold greater than the distilled deionized water added in step 9 Add 5 1 wash additive use only 5 1 wash additive regardless of the original sample volume Vortex until the pellet 1s fully dispersed Note The protein pellet will not dissolve in the wash buffer 11 12 13 14 15 Incubate the tubes at 20 C for at least 30 min Vortex for 20 30 s once every 10 min At this stage the tubes can be stored at 20 C for up to one week with minimal protein degradation or modification Centrifuge the tubes at 8000 _ for 10 min Carefully remove and discard the supernatant A white pellet should be visible Allow the pellet to air dry briefly no more than 5 min Note Do not over dry the pellet If it becomes too dry it will be difficult to resuspend Resuspend each pellet in rehydration solution for first dimension The volume of rehydration solution used can be as little as 1 20 of the volume of the original sample See next section for examples of rehydration solutions and volumes appropriate for different applications Vortex the tube for 30 s Incubate at room temperature Vortex or aspirate and dispense using a pipette to fully dissolve Note If the pellet is large or too dry it may be difficult to resuspend fully Sonication can speed r
30. ide Mass Fingerprints PMFs Query results inl x r Move Protem Summary Report Back F d Format As Protein Summary Help cignificance threshold p 0 05 number of hits Overview Table Results Clear Get All Get Hit s r Click on column header to Jump to entry m results list Mowe mouse over any mdicater to highlight identical peptides Click on an indicator to see details of individual match Use check boxes to select sub set of queries for new search IvInuse over AAN17824 LVNELTEFAK Hit 1 2 3 4 5 6 7 8 9 10 m 820 49 e eee m 906 49 9 9 9 jo 922 4804 9 9 9 jo iM 9245009 9744505 m 987 55 joo 2 101754 0 11426604 1145 63 19 Q 163 60 09 o E al HE Figure 6 5 Overview table of matching peptides Further down the page under Index there is a summary of the result with scores and sequences The molecular weight of the proteins is often useful to tell false positives due to either excessively high gt 300 kDa or very low 5 kDa molecular weights On this level you may select the entries you would like to visualize within BioTools 6 8 Tutorials for BioTools Version 3 2 Bruker Daltonik GmbH Define the Search Qu
31. ility than analyte molecules and crystallize closer to the surface of the preparation Preparations of water insoluble matrices can therefore be washed with e g 0 1 TFA Salts will more readlily dissolve and improved signal noise ratios can be obtained Washing is usually performed by applying 1 5 ul of washing solution 0 1 TFA Water on top of the preparation waiting for a few seconds and removing the droplet by pipetting or by filter paper One should carefully avoid to touch the crystalline surface of the preparation If loss of sample during washing 15 a concern typically with thin layer preparations the use of chilled washing solution is recommended Recrystallisation If the sample contains a high amount of salt the spectra qualitiy can be further improved by recrystallisation of the spot after washing Recrystallisation 15 performed by applying a small volume of organic solvent TA mixture in most cases Thin layer and double layer preparations will lose their specific advantages Also it may be possible to reduce the quality of the spectra especially in the case of low amount of sample For that reason it is recommended to first measure the washed sample and perform the recrystallisation only after no satisfying spectra could be obtained washed q i directly prepared id lial 0400 400 _ 1600 mf salty sample direct preparation green and after on targe
32. le only for HCCA The matrix 15 prepared on the target to form a thin layer of very small and homogenous crystals This is achieved by dissolving the matrix in Acetone After spotting this solution on the target the acetone spreads on the target and evaporates very fast The thin matrix layer remains on the surface of the target The sample 1s applied on top of this thin layer After the sample is dried the analyte molecules remain on top of the matrix Advantages of the thin layer method are the very homogenous size of the crystals The methods yields high resolution spectra and the detection limit is increased 13 compared to the dried droplet method Thin Layer preparations can be washed to remove salts but compared to the dried droplet method there 15 a higher possibility to remove also analyte molecules If the sample 15 too basic there is also a possibility that the thin layer is dissolved Thin layer preparations can be recrystallized but then all specific advantages of this preparations are lost One significant disadvantage of thin layer preparations are the very limited number of laser shots that can be applied at one sample position Usually after 10 to 20 laser shots no spectra can be acquired anymore This limits especially the usability of thin layer preparation for PSD Experiments The Thin Layer Method can be enhanced by preparing a thin layer containing nitrocellulose This retains peptides more efficiently 1 the washing step Th
33. lease refer to the SequenceEditor Tutorial Protein Digests chapter P 5 1 Search for Unexplained Masses after MASCOT search Problem 2 Sequence coverage is too poor after import of a search result Assumption 1 or a script may have missed picking the weak peaks in the spectrum so far and need to find out do a theoretical digest of the identified protein and send the predicted masses to the spectrum Then add the missed peaks to the peaklist please refer to the SequenceEditor Tutorial Protein Digests chapter P 2 Perform Enzymatic Digest chapter P 3 Format the Digest Results and chapter P 4 Export Digest Results to Spectrum Assumption 2 need to do an LC ESI MS MS run for better coverage and want to set up a preferred or exclusion mass list Do a theoretical digest of the identified protein and export the predicted m z values to _ esguireControl please refer to the SeguenceEditor Tutorial Protein Digests chapter P 2 Perform Enzymatic Digest chapter P 3 Format the Digest Results and chapter P 4 Export Digest Results to Spectrum Tutorials for BioTools Version 3 2 6 17 Bruker Daltonik GmbH Sequence Database Searches from MALDI Peptide Mass Fingerprints PMFs Problem 3 A particular peak remains unaccounted for in the mass fingerprint after all my efforts and I really want to Know what it is Run an MS MS spectrum LIFT CID etc first and try a library search in any case with that spectrum even without enzyme spe
34. n period Enzyme concentration and activity depends upon the specification of the enzyme Enzyme specification can be obtained from the manufacturer manual Enzyme specifications a Source of enzyme b Optimum temp pH for enzyme activity Conc of CaCl in buffer 1f required TPCK treated Enzyme specificity low or high Enzyme grade Sequencing grade etc Trypsin SIGMA 20ng ul re separaUon eparduor OF Proteins igestot Proteins of Pepudes FPLC Freeflow Electrophoresis 1D Gel Electrophoresis 20 Gel Electrophoresis 1D HPLC 2D HPLC 1D HPLC 2D HPLC Capillary Electrophoresis y PA cyd Nano LC MALDI TOF TOF Setup 8 S 8 155 3 so MEE BE BE p Pz KB HE i D Data Tutorial Data Bioto no EI M amp beta casein precursor E M Digest Matches Score Search Parameter Ch l i MSMS 1 MSMS 1 262 145 y1 y2 y 16 503 300 a8 147 208 y4 2061 972 y1 747 301 977 547 1315 466 ds 390 252 y5 y6 y a8 b10 1559 827 a y2 y3 b12 y14 2043 791 632 297 876 390 1186 376 ai 120219 276 232 y7 1430 456 1786 307 1 b2 hy b11 y14 i 2 200 400 600 800 1000 1200 1400 1600 1800 2000 miz Proteir ew Match Errors MS MS Fragments MSMS Analysis Mods
35. onstant shaking on a shaker followed by an additional 15 20 mins incubation using a fresh equilibration buffer supplemented with 135mM iodoacetamide instead of DTT SECOND DIMENSIONAL SEPARATION The second dimensional separations were carried out on 12 SDS polyacrylamide gels according to the procedure previously reported by Laemmli The gel was then stained with Coomassie Brilliant Blue R 250 for two hours minimum and destained with destaining solution Methanol acetic acid water Rehydration buffer RB Urea 7M Thiourea 2M if required CHAPS 4 Ampholytes Or Pharmolytes 0 2 0 5 Aliquotes of RB without adding DTT can be stored at 20 C Methodology for InGel Digestion Coomassie Stained with Trypsin or Chymotrypsin 1 Excision of protein bands from Polyacrylamide gels 1 The detained gel was washed with MQ H2O for 2X 10 mins The protein bands of interest were cut using sterile scalpel or 100 ul pipette tip and was transferred into a fresh eppendorf pre rinsed with 100 ACN and air dried 2 Washing and Equilibration 1 gel pieces were washed with 100 500 ul MQ depending upon the size of gel pieces for 3 X 5 mins at RT with gentle agitation on vortex mixer low speed They were then equilibrated with 100 ul of 100 mM Ammonium bicarbonate buffer for 20 mins at RT with gentle agitation ii NH4HCO3 buffer was then discarded and gel pieces were washed with 1 1
36. oteomics projects is the identification of a protein sample based on an endoprotease digest such as trypsin and a sequence database search using the m z values of the digested peptides Such data are called peptide map or peptide mass fingerprint BioTools allows such searches to be performed on all available search engines Internet access provides and particularly operates in a seamless way with the MASCOT search program Matrix Science Ltd London up to the latest version A prereguisite is a spectrum with annotated monoisotopic masses either from FlexAnalysis FLEX or DataAnalysis Trap oTOF FTICR In FlexAnalysis the method PMF FAMSMethod performs a peak finding process algorithm SNAP in the mass range of 800 4000 m z In order to eliminate background peaks just click on MassList Filter Background Peaks and then choose a suitable MassControlList containing the most common background masses for trypsin autoproteolysis or contaminants like keratins Tutorials for Bio Tools Version 3 2 October 2008 6 1 Bruker Daltonik GmbH Sequence Database Searches from MALDI Peptide Mass Fingerprints PMFs 6 2 Sequence Database Search using a MALDI Peptide Mass Fingerprint Sequence Database Search using a MALDI Mass Fingerprint An example PMF dataset is in the tutorial data directory Please follow the described steps Open the tutorial data set utorial Data Biotools Flex BSA_digest O_G11 1 1SRef pdata
37. otools Tutorial data Flexi BSA digest LysC 1sref pdata 1 1R a B Bl l File Edit View Analysis Search Tools Window Help la x co ed iB mi amp x Ra m rs E ws so EAR 8 Teehemchy Meas Cale MH mt Range P Sequence D data Biotools Tutorial data Flex BSA digest LysC LysC digest von BSA 4 Unmatched AM ih Peaks peak 4 924 503 peak 8 1017 542 9 1142 664 peak 13 1347 521 peak 16 1917 063 peak 18 2435 159 peak 19 2553 121 peak 20 2554 118 peak 21 2555 144 Aw AF542068 NID Bos taurus AAN17824 See J lii Digest Matches Score 190 00 Search Parameter Charge 1 MS Tol 50 00 ppm j peak 1 820 487 820 468 0 020 229 235 j 906 490 906 472 0 018 205 211 922 477 922 467 377 0 010 373 205 211 974 447 974 458 0 010 37 44 987 546 987 538 0 009 29 36 1014 607 1014 620 0 012 549 557 1145 630 1145 643 0 013 236 245 1163 605 1163 631 0 026 66 75 1305 695 1305 717 0 021 402 412 1399 697 1399 693 0 005 569 580 1897 073 1897 076 0 002 438 455 2028 093 2028 103 0 009 421 437 j peak 22 2953 386 2953 504 0 118 161 183 FGERALK IETMREK IETMREK 4 Oxidation DLGEEHFK SEIAHRFK QTALVELLK AWSVARLSOK Intensity coverag LVNELTEFAK HLVDEPONLIK TVMENFVAFVDK VPDVSTPTLVEVSRSLGK LGEYGFONALIVRYTRK YLYEIARRHPYFYAPELLYYANK
38. problems elimination of some masses which are shared by isobaric peptides from the different proteins may prevent the search engine from identifying the 2nd protein Tutorials for BioTools Version 3 2 6 5 Bruker Daltonik GmbH Sequence Database Searches from MALDI Peptide Mass Fingerprints PMFs 6 3 1 MASCOT Search Results The basic information in the result header is the top score its access number in the database and the entry name followed by a histogram representation of the 50 top scores Protein Summary Report Within the green rectangle the likelihood of a false positive match is 5 or more Usually only scores significantly outside this region Scores gt 70 are significant Good values are gt 100 Attention The absolute score for the 5 false positive likelihood is a function of the database and the search conditions It may vary To continue with importing the top hit into BioTools press the Get Hit s button and continue reading in section 6 3 3 Work with Search Results in BioTools Query results 15 x Move MATRIX cence Mascot Search Results _ Forward User Email beGhdal de Results Search title Database MSDB 20040630x 1501893 sequences 480537664 residues Elear Taxonomy Other mammalia 31680 sequences Get All Times t amp 10 Sep 2004 at 11 45 12 GHT Top Score 190 for AAA51411 B VALBUMIH HID Bos taurus Get r Probability Based Mowse S
39. s the minimum amount needed for analysis is 16 picomoles microliter The minimum for mw 20 000 or less 15 60 picomoles microliter For 66 000 mw the minimum amount needed for analysis 1s 160 picomoles microliter Therefore the larger the molecular weight the more sample is needed Give information like structure sequence molecular weight type of compound biological activity chemical reactivity pH sample amount concentration describe purification isolation with focus on relative agents solvents known or suspected impurities suitable solvents hazardous properties radioactivity carcinogenicity poison or explosive 10 Sample Preparation Aims of the sample preparation The ideal sample preparation in MALDI would be a homogenous layer of small matrix crystals containing a solid solution of the analyte To obtain the best result there 1 a choice of different matrices as well as preparation techniques Both choices depend on the nature of the analyte One aim 15 to obtain as homogenous preparation of the matrix both in terms of sample distribution and term of the sample geometry The following picture illustrates the effects of a matrix preparation with small homogenous crystals compared to a preparation containing crystals of different sizes U U U E j m z U
40. s it can be a major source of frustration due to failed identifications if the error estimation was a bit too optimistic So be sure about your data quality and evaluate the quality with a simple rule Rule of thumb for every 200 Da monoisotopic integer molecular weight add 0 1 Da oo at MW 1000 expect as average 1000 5 as exact mass at MW 2000 exact mass is 2000 0 Using this rule it is easy to estimate the correctness of your calibration The exact rule is Am 1 00048 INT m Matthias Mann 43 ASMS Conference 639 Providing you with the expected first decimal for any peptide ion mass 200 1 400 2 600 3 800 4 1000 5 etc Typically the precursor Protein mass does not need to be specified However the largest possible mass can be specified here e g known from gel analysis to restrict the retrieval of unspecific matches from extremely large proteins For each database entry Mascot looks for the matching peptides which are within a contiguous stretch of sequence less than or equal to the specified protein molecular weight This will often be less than the mass of the entire sequence entry unless the data set happens to include both the N terminal and C terminal peptides The number of missed cleavages or partials accounts for tolerated internal missed cleavage sites in matching peptides This number should be set to 0 or 1 since higher values reduce the specificity of the search as extensive use of variable modifi
41. ss F1 NUM suckau X 1636 2 m z Y 4807 52 Abs Int Figure 6 1 BSA tryptic digest Lys C from ultraflex Il The fully annotated spectrum appears resulting from a previous MASCOT search The following description allows you to generate this information yourself 6 2 Tutorials for BioTools Version 3 2 Bruker Daltonik GmbH Define the Search 6 3 Define the Search Push the 95 button to open the MS data search dialog Note connection to a MASCOT Inter or Intranet server cannot be established an empty search dialog pops up after a few seconds The local address is of the type http lt servername or IP address gt mascot cgi nph mascot exe 1 must be added to the URL list to do local searches In addition the perl scripts from the BioTools installation CD should be installed on local Mascot server older than version 2 1 If the Internet address could not be reached you need to setup the Internet connection Peptide Mass Fingerprint 7 x http www matrixscience com cgi nph mascot exe 71 Setup Matris Science home page User Mame Email Search Title Other mammalia Database SwissProt Enzyme Trypsin Global Acetyl K 4 Variable Modifications Acetyl M term Modifications 018 Amide L term Biotin K Oxidation H FPF Protein M gt kDa Missed Lleavages lt 2
42. sults window The viewer is directly linked to the tree view as well as the spectrum which means they all together display information about the same set of peptides within the same downloaded sequence The matching peptides are represented here as bars underneath the covered sequence range which allows you to visualize the information extracted from the spectrum on the sequence level The view can be configured using the pull down menu opened by right mouse button click in context with the sequence viewer 33 BioTools D Data Biotools Tutorial datasFlexBSA digest M8s1 1SRefpdatas1 s1r S iol xj la xj File Edit View Analysis Search Tools Window Compass Help a Ed dB BLA amp Be m zs s so M F 15 W Bv Tree hierarchy fA D Data Biotools T utorial data Flex B H E Unmatched cu 1888 924 V j peak 7 1479 792 1567 742 169 183 B P02763 Serum albumin precurs 421 433 347 359 Digest Matches Score 113 00 1880 927 1 508 523 Abs Int 1000 1724 840 469 482 2045 028 168 183 1362 657 89 100 1283 694 361 371 1163 601 66 75 8 88 0 MEXIMIS 1250 Protein View Match Errors MSHS frecments MSHS Analysis 1667 809 469 482 1639 943 437 451 1500 1750 1823 897 508 523 1749
43. t wash blue sodium adducts are marked with Signal to Noise ratio is improved after washing while sodium adduct formation is decreased E 15 WARP LC Software ool for ProteineerL Workflows VWAKP LC Integrated Software Tool V V orkflow dministration Dy R esult driven rocessing for LC based approaches Sample Handlinc FLEXCONTROL Graphical User Interface GUI A TOFITOF Pepmix par ee mee SS amp 112 TN K Button Bar dd ee dal TES E fea E L 1 11 wei A en aes j MT ae e Da 100 AT RA REL Le N Wy WWE aie ee Ue Sy 308331 1614509 E fu T i TW Z o Nn Hw CIA Shed min q 32 ru 5 z elt FAR Lunar Spectoreste Fermo Test Sab hur V pss z Bpample Carrier Cl shoe ese Com T_T 10 FF Faris 2EF For pran F1 19 FLEXANALYSIS D
44. taurus Cr5pecies 824 Bos taurus nin 4 Fe _ j_ __ F Emi uma 2 Figure 6 8 Error view 6 3 3 Work with Search Results in BioTools If you decided to import candidate sequence 1 from the MASCOT Query results window for further work in BioTools push the Get Hit s button in the Ouery results window with 1 in the entry field below If you would like to import e g entries 1 4 and 8 specify 1 4 8 If you like to import all entries push the Get All button but it is recommended not to do this since the download of all seguences may cause long waiting times In particular if a database is accessed via the web and the total number of hits was selected to be gt 10 To avoid importing too many redundant protein seguences Mascot 2 0 guery results are best viewed after Concise View formatting Older versions of Mascot only provide the Protein view Tutorials for BioTools Version 3 2 6 11 Bruker Daltonik GmbH Sequence Database Searches from MALDI Peptide Mass Fingerprints PMFs gt i Query results D xj Move Back ae MATRIX sciences Mascot Search Results User Email be bdal de Resulta Search title Database M5DB 20040630 1501893 sequences 480537664 residues __ Taxonomy Other mammalia 31680 sequences Get All Time t amp 10 Sep 2004 at 11 45 12 Top Score 190 for AAASI411 BOVALBUMIN HID Bos taurus Get Hit s Prob
45. volume of sample add three volumes of precipitant Mix well by vortexing or inversion Incubate on ice 4 5 C for 15 min For each original volume of sample add three volumes of co precipitant to the mixture of protein and precipitant by vortexing briefly Position the tubes In a microcentrifuge with the cap hinges facing outward Centrifuge at 8000 _ 2 for 10 min Remove the tubes from the microcentrifuge as soon as centrifugation has finished A pellet should be visible Proceed rapidly to the next step to avoid resuspension or diffusion of the pellet Remove as much of the supernatant as possible by decanting or careful pipetting Do not disturb the pellet Carefully position the tubes in the microcentrifuge with the cap hinges and pellets facing outward Centrifuge the tubes for at least 1 min to bring any remaining liquid to the bottom of the tubes Use a pipette to remove the remaining supernatant There should be no visible liquid remaining in the tubes To each tube add three fold to four fold more co precipitant than the size of the pellet Carefully reposition the tubes in the microcentrifuge with the cap hinges facing outward Centrifuge for 5 min Use a pipette to remove the supernatant Pipette enough distilled or deionized water on top of each pellet to cover the pellet Vortex each tube for several seconds The pellets should disperse but not dissolve in the water Add 1 ml of wash buffer pre chilled
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