Rapid Assay Development and Refinement forTargeted Protein Quantitation Using anIntelligent SRM (iSRM) WorkflowReiko Kiyonami1, Alan Schoen1, Amol Prakash1, Huy Nguyen1, Scott Peterman1, Nathalie Selevsek2, Vlad Zabrouskov1, Andreas Huhmer1, Bruno Domon21Thermo Fisher Scientific, San Jose, CA; 2ETH Zurich, Switzerland

IntroductionSelected reaction monitoring (SRM) using triplequadrupole mass spectrometers has been the foundationfor small molecule quantitation for several decades. It isalso emerging as a powerful technique for targetedquantitative proteomics due to its high sensitivity,selectivity, and throughput1,2. However, developing anSRM assay for targeted proteins remains challenging. Oneof the main obstacles is finding the best peptides that aresequence specific or unique and give sufficient signalintensity without using specific protein/peptide standards.Peptide selection generally relies on using either priorproteomics discovery data or computational prediction3. Acandidate peptide list can be rather long to cover multipleproteins of interest and the required transition list caneasily exceed one thousand. To quickly find the bestprototypic peptides and the best SRM transitions for eachpeptide from the large number of candidates, a triplequadrupole system must be able to survey hundreds ofpeptides, with proof of their identities, in a single HPLC-MS run.

To address this challenge, we’ve incorporated a newintelligent SRM (iSRM) technology into the currentThermo Scientific TSQ family of triple quadrupole massspectrometers for quantitative proteomics. Thistechnology enables processing of over 15,000 SRMtransitions for simultaneous quantification and identityconfirmation of up to 1000 peptides in a single HPLC-MSrun4, which dramatically reduces the labor and cost ofdeveloping multiplexed SRM assays. It is fully supportedby our Pinpoint software5 (www.thermo.com/pinpoint)from iSRM instrument method generation to dataprocessing.

In this paper, we demonstrate use of Thermo ScientificPinpoint software and the iSRM workflow to develop ahighly multiplexed SRM assay to simultaneously quantifyand identify forty yeast proteins.

GoalDemonstrate the utility of iSRM technology by developingand refining an iSRM workflow for the simultaneousanalysis of 40 proteins. Then, use the iSRM workflow toidentify and quantify those proteins in a single analysisand assess the precision of the resulting measurements.

Experimental Conditions

Sample preparationA whole yeast cell lysate was solvated in 0.1 Mammonium bicarbonate buffer containing 8 M urea at afinal concentration of 1 mg/mL. The yeast cell lysatesolution was reduced, alkylated and enzymaticallydigested. 40 yeast proteins (Table 1) found in a previousdiscovery experiment using a Thermo Scientific LTQOrbitrap hybrid mass spectrometer were targeted.

Key Words

• Targeted proteinquantitation

• iSRM

• Pinpoint

• TSQ Vantage

• Triple quadrupole

ApplicationNote: 468

# Protein1 >gi|6320359|ref|NP_010439.1| Cpr1p [Saccharomyces cerevisiae]2 >gi|27573538|pdb|1F18|A Chain A3 >gi|6322790|ref|NP_012863.1| Fba1p [Saccharomyces cerevisiae]4 >gi|6324951|ref|NP_015020.1| Gdh1p [Saccharomyces cerevisiae]5 >gi|14318506|ref|NP_116640.1| Hsp12p [Saccharomyces cerevisiae]6 >gi|6324594|ref|NP_014663.1| Hsp10p [Saccharomyces cerevisiae]7 >gi|6322225|ref|NP_012300.1| Lys1p [Saccharomyces cerevisiae]8 >gi|124875|sp|P00817|IPYR_YEAST 9 >gi|6321523|ref|NP_011600.1| Pil1p [Saccharomyces cerevisiae]10 >gi|6323840|ref|NP_013911.1| Cytoplasmic chaperone of the Hsp90 family11 >gi|4195|emb|CAA26184.1| unnamed protein product [Saccharomyces cerevisiae]12 >gi|6324151|ref|NP_014221.1| Protein component of the small (40S) ribosomal subunit13 >gi|6325176|ref|NP_015244.1| Protein component of the small (40S) ribosomal subunit14 >gi|7245976|pdb|1QPB|A Chain A15 >gi|10383781|ref|NP_009938.2| 3-phosphoglycerate kinase16 >gi|6319546|ref|NP_009628.1| Hsp26p [Saccharomyces cerevisiae]17 >gi|6320936|ref|NP_011015.1| Cobalamin-independent methionine synthase18 >gi|6324212|ref|NP_014282.1| Malate synthase19 >gi|6322815|ref|NP_012889.1| UDP-glucose pyrophosphorylase (UGPase)20 >gi|6323335|ref|NP_013407.1| Aco1p [Saccharomyces cerevisiae]21 >gi|6322323|ref|NP_012397.1| Tif2p [Saccharomyces cerevisiae]22 >gi|6321772|ref|NP_011848.1| Protein component of the small (40S) ribosomal subunit;23 >gi|6324291|ref|NP_014361.1| Idh1p [Saccharomyces cerevisiae]24 >gi|6324950|ref|NP_015019.1| Ald4p [Saccharomyces cerevisiae]25 >gi|6322066|ref|NP_012141.1| Component of the mitochondrial alpha-ketoglutarate dehydrogenase

complex which catalyzes a key step in the tricarboxylic acid (TCA) cycle26 >gi|6319972|ref|NP_010052.1| Cytoplasmic ATPase27 >gi|6321968|ref|NP_012044.1| Eno2p [Saccharomyces cerevisiae]28 >gi|3793|emb|CAA27203.1| unnamed protein product [Saccharomyces cerevisiae]29 >gi|6325253|ref|NP_015321.1| Lsp1p [Saccharomyces cerevisiae]30 >gi|6324328|ref|NP_014398.1| Cit1p [Saccharomyces cerevisiae]31 >gi|6323288|ref|NP_013360.1| Hsp60p [Saccharomyces cerevisiae]32 >gi|487960|gb|AAB68391.1| Yhr064cp [Saccharomyces cerevisiae]33 >gi|6324343|ref|NP_014413.1| Acc1p [Saccharomyces cerevisiae]34 >gi|6320710|ref|NP_010790.1| S-adenosylmethionine synthetase35 >gi|6323387|ref|NP_013459.1| Acetohydroxyacid reductoisomerase36 >gi|6321013|ref|NP_011092.1| Pab1p [Saccharomyces cerevisiae]37 >gi|3724|emb|CAA24608.1| unnamed protein product [Saccharomyces cerevisiae]38 >gi|6321679|ref|NP_011756.1| Alpha subunit of heterooctameric phosphofructokinase39 >gi|24158771|pdb|1KB9|A Chain A40 >gi|114589|sp|P05626|ATPF_YEAST ATP synthase subunit 4

Table 1: 40 yeast proteins found in a previous discovery experiment

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LC/MS

Nano-HPLC

Pump: nano-LC 1D plus, EksigentColumn: PicoFrit C18 column from New Objective

(50 µm x 100 mm, 10 µm tip)Flow rate: 300 nL/minBuffer A: Water containing 0.1% formic acidBuffer B: Acetonitrile containing 0.1% formic acidGradient: 5% B to 45% B in 40 minSample loading: directly loaded on columnInjection amount: 1 µL

MS

Thermo Scientific TSQ Vantage equipped with a nanospray sourceCapillary temperature: 200 °C; Spray voltage: 1800 ViSRM set up (primary SRM and data-dependent SRM scans):

Two primary and additional six secondary fragment ions per peptideQ1 & Q3: 0.7 FWHM DaQ2: 1.2 mTorr (Ar)Cycle time: 2.0 sOne time window of 50 min for all the transitions for the initial iSRM runs4 min window per transition for the refined iSRM runsCollision energy: 0.034 x precursor ion mass m/z + 3.314

Dynamic exclusion:Intensity threshold: 300Scan time: 0.1 s (100 ms)Repeat count for triggering data-dependant SRM scan: 5Exclusion time: 20 s

iSRM workflow logicThe iSRM workflow utilizes SRM specificity in two waysfor simultaneous quantification and identity confirmationof targeted peptides. The first is compound-specificquantification using a time-based SRM acquisition with aconstant cycle time that monitors several primarytransitions for each peptide. The second is a data-dependent SRM acquisition that monitors both thoseprimary and additional secondary transitions for peptidesequence confirmation. The data-dependent SRMacquisition is triggered only when the intensities of allprimary SRM transitions simultaneously exceed eachindividually defined intensity threshold for a presetnumber of cycles. The unique dynamic exclusion designallows each secondary scan to be triggered only once foreach LC peak. The goal is to trigger the data-dependantSRM around the apex of each peak by setting the repeatcount equal to half of the LC peak width divided by thecycle time. The dynamic exclusion allows acquisition ofsufficient structural information to unambiguouslyconfirm the compound’s identity without significantlyperturbing the quantification obtained with the primarySRM transition list. This prevents the user from wastingprecious acquisition time and increases the overallproductivity and throughput of the experimentdramatically. Figure 1 shows the flowchart of the iSRMworkflow logic.

Figure 1. Flowchart of the iSRM workflow logic

iSRM workflow development processThe iSRM workflow development process is iterative.Target proteins found through discovery experiments areimported into Pinpoint™ software. The Pinpoint softwarelargely automates the preliminary selection of primary andsecondary transitions to be monitored based on thediscovery experiment results. The resulting list of peptidesand transitions is used in a preliminary iSRM method. Asample containing the targeted peptides is run using thepreliminary method and the resulting data is importedinto the Pinpoint software. Pinpoint software providestools for evaluating the data. The target peptides thatshow the best responses are retained while target peptidesthat do not show good responses are removed from thefinal iSRM method. This ensures that the limited, fixedcycle time is used to analyze only peptides that providehigh-quality results.

Results

Initial peptide/transition selection for generating theiSRM instrument method based on the proteomicsdiscovery search resultsA list of forty targeted proteins that was generatedthrough proteomics discovery experiments was importedinto Pinpoint software (Figure 2). The spectral librarymanagement option was used to add the discovery searchresults as a spectral library. All the pre-detected peptidesfrom each targeted protein were selected automatically.

Batch mode was used to automatically selecttransitions. Based on signal intensity from the spectrallibrary, two primary (most intense) and six secondarytransitions (in the order of intensity) were selected foreach peptide. This yielded a total 2952 transitions (738primary and 2214 secondary) for the 369 selected peptides(Figure 3). The yielded 2952 transitions were furtherexported to csv file for generating the initial iSRMinstrument method.

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Figure 2. Initial peptide selection based on previous proteomics discovery data

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Figure 3. Automatic transition selection based on the MS/MS spectral library

Figure 4. Export initial transitions to a .csv file for generating the preliminary iSRM method

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It is critical in this exporting step to estimate the dwelltime per primary transition based on the equation:

(cycle time – total preset time of primary transitions)/totalnumber of primary transitionsThis was done to ensure the dwell time was long enough(≥ 2ms) to achieve required sensitivity and reproducibility.Computation using a two-second cycle time, one-millisecond preset time, and a single 50-minute timewindow for all primary transitions yielded an estimateddwell time of

Data analysis and scoring for evaluating the initialpeptides/transitions selection and refining the method tobuild the final iSRM assayThe initial main workbook file was opened in the Pinpointsoftware and the two raw files were added for dataprocessing. Pinpoint software stitched the two initial runsback together for data processing without manualintervention.

The color filter management tool was used to analyzethe peptides based on the user-defined correlation p-valueand library spectra filters. The peptides that gave p valuesless than 0.05 were considered confidently sequenceconfirmed and presented as green. The peptides that gavep values larger than 0.05 were considered not sequenceconfirmed and presented as yellow. The undetectedpeptides were presented as white (Figure 6).

The peak shape of early eluting green peptides wereevaluated and the peptides that exhibited bad peak shapewere removed using the “Remove retention time for thecurrent selection” tool in the Data Analysis Managementmenu. A refined peptides/transitions list was built byretaining only the high-confidence green-coded peptides

with good peak shapes (Figure 7). The Pinpoint softwarescheduled the time window of each peptide automaticallybased on the retention time and user-defined schedulingwindow size (±2 min) for identified peptides.

The refined .csv list was exported to generate a finaliSRM assay that targeted 153 peptides, yielding a total of1224 transitions (306 primary and 918 secondary). Thereduced number of peptides and transitions and the tightscheduled time windows in the final method makes itpossible to run future samples in a single analysis withsufficient dwell time for each primary transition.

The simultaneous quantitative and qualitative results ofthe selected 153 peptides (representing the 40 proteins)using the refined final iSRM methodThe final iSRM method for targeting 153 yeast peptides(40 proteins) was used to analyze the yeast sample inquintuplet in order to assess analytical precision of thecomplete workflow. The integrated peak areas of twoprimary transitions per peptide were used for thequantification and the correlation p-values between thecomposite MS/MS spectra and the library spectra wereused for peptide identification.

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Figure 6. Peptide sequence verification and quantitation from the initial SRM assay

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Figure 7. iSRM method refinement based on data acquired with the preliminary iSRM method

The final iSRM method successfully confirmed theidentity of each eluted peptide by matching the compositeMS/MS spectra to the MS/MS library and collected precisequantitative data for each identified peptide. Theanalytical precision was excellent (Figure 8). Ninety twopercent of the targeted peptides had %CVs ≤5%. Ninetynine percent of the targeted peptides had %CVs below10% and all the peptides had %CVs below 12% (Table 2).

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Figure 8. Peptide sequence verification and quantitative precision from the refined iSRM assay

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Peak Area Peak Area Peak Area Peak Area Peak Area# Protein Peptide RT n=1 n=2 n=3 n=4 n=5 CV%1 >gi|6320359|ref|NP_010439.1| Cpr1p [Saccharomyces cerevisiae] 16018500 16393800 17042400 17284100 17250500 3

GFGYAGSPFHR 24.55 673700 680200 725300 671100 645600 4LYNDIVPK 21.72 13390000 13850000 14350000 14620000 14660000 3VESLGSPSGATK 12.77 1530000 1474000 1536000 1554000 1535000 2VIPDFMLQGGDFTAGNGTGGK 33.1 424800 389600 431100 439000 409900 4

2 >gi|27573538|pdb|1F18|A Chain A 1431680 1377960 1474900 1460780 1464890 3FEQASESEPTTVSYEIAGNSPNAER 25.59 1411000 1359000 1454000 1437000 1439000 2GSFKDSLIK 20.36 20680 18960 20900 23780 25890 11

3 >gi|6322790|ref|NP_012863.1| Fba1p [Saccharomyces cerevisiae] 2296340 2466560 2504950 2508200 2517800 4GAIAAAHYIR 19.71 2036000 2201000 2232000 2207000 2250000 4SPIILQTSNGGAAYFAGK 28.85 218100 225600 229800 253800 229100 5TGVIVGEDVHNLFTYAK 33.4 42240 39960 43150 47400 38700 7

4 >gi|6324951|ref|NP_015020.1| Gdh1p [Saccharomyces cerevisiae] 5770160 5817680 5944640 6129130 6250570 3AANLGGVAVSGLEMAQNSQR 29.86 174300 165700 162600 163200 160000 3FIAEGSNMGSTPEAIAVFETAR 34.21 187600 162900 184500 193300 189000 6GANIASFIK 26.89 2425000 2487000 2506000 2670000 2765000 5HIGQDTDVPAGDIGVGGR 22.44 68560 71880 68440 76730 72970 4NSLTGLDMGGGK 21.9 2758000 2782000 2868000 2858000 2910000 2SLEQIVNEYSTFSENK 34.66 156700 148200 155100 167900 153600 4

5 >gi|14318506|ref|NP_116640.1| Hsp12p [Saccharomyces cerevisiae] 10145560 10022570 10543250 10363100 10324950 2DNAEGQGESLADQAR 18.11 577400 596100 609800 599000 591300 2GKDNAEGQGESLADQAR 17.01 221700 206500 222000 197700 208300 4GVFQGVHDSAEK 17.41 2054000 1874000 1998000 1801000 1864000 5LNDAVEYVSGR 21.71 7219000 7280000 7644000 7695000 7590000 3SYAEQGKEYITDK 20.01 73460 65970 69450 70400 71350 4

6 >gi|6324594|ref|NP_014663.1| Hsp10p [Saccharomyces cerevisiae] 2125500 2149100 2223700 2257000 2206000 2LGNDDEVILFR 29.81 938500 961100 989700 1017000 1004000 3VGDQVLIPQFGGSTIK 31.1 1187000 1188000 1234000 1240000 1202000 2

7 >gi|6322225|ref|NP_012300.1| Lys1p [Saccharomyces cerevisiae] 286370 270440 285580 310660 300970 5IYVEDSPQSTFNINEYR 29.51 81870 65540 77380 85360 83970 9QAGAIIVPAGSWK 27.28 204500 204900 208200 225300 217000 4

8 >gi|124875|sp|P00817|IPYR_YEAST 6987000 7064900 7298800 7377000 7011300 2EETLNPIIQDTK 24.29 537000 524900 546800 559000 536300 2GIDLTNVTLPDTPTYSK 30.75 1314000 1378000 1444000 1417000 1368000 3LNDIEDVEK 18.55 3827000 3859000 3977000 4076000 3792000 3VIAIDINDPLAPK 31.74 1309000 1303000 1331000 1325000 1315000 1

9 >gi|6321523|ref|NP_011600.1| Pil1p [Saccharomyces cerevisiae] 4824200 4733400 4867900 5183800 5198800 4ALLELLDDSPVTPGETRPAYDGYEASK 35.29 165700 153800 182900 174800 167800 6IALIAGYGK 24.37 1052000 1065000 1123000 1137000 1192000 5IEVLEQELVR 28.07 999500 985600 1063000 1090000 1108000 5SAAGAFGPELSR 22.43 2607000 2529000 2499000 2782000 2731000 4

10 >gi|6323840|ref|NP_013911.1| Cytoplasmic chaperone of the Hsp90 family 9072600 9467100 9661000 10090600 9827900 4AILFIPK 28.26 3170000 3495000 3617000 3759000 3736000 6DSSMSSYMSSK 18.25 760900 784200 792300 758300 788700 2GVVDSEDLPLNLSR 29.22 4053000 4145000 4169000 4461000 4221000 3NPSDITQEEYNAFYK 27.29 232400 226400 233900 256800 229500 5TGQFGWSANMER 25.75 856300 816500 848800 855500 852700 2

11 >gi|4195|emb|CAA26184.1| unnamed protein product [Saccharomyces cerevisiae] 6291700 6424900 6711100 6787000 6555100 3DGPLSTNVEAK 17.7 1288000 1358000 1353000 1373000 1295000 3LEFANLTPGLK 30.29 3171000 3204000 3388000 3405000 3280000 3NELITSLTPGVAK 27.8 1549000 1584000 1672000 1721000 1702000 4YAMALSYFAK 30.71 283700 278900 298100 288000 278100 3

12 >gi|6324151|ref|NP_014221.1| Protein component of the small (40S) ribosomal subunit 7941100 8006300 8250900 8493800 8580700 3ELAEEGYSGVEVR 22.54 741400 780000 804300 817900 833900 4GLSAVAQAESMK 22.92 1326000 1330000 1401000 1398000 1396000 3INELTLLVQK 29.68 543300 564100 575800 590000 629100 5LLNGLAIR 26.1 4504000 4407000 4531000 4731000 4771000 3YAPGTIVLYAER 28.15 826400 925200 938800 956900 950700 5

13 >gi|6325176|ref|NP_015244.1| Protein component of the small (40S) ribosomal subunit 12995300 13248500 12879000 14159000 14296100 5LDYVLALK 28.56 215300 222500 243000 246000 256100 6LFEGNALIR 26.58 6831000 6803000 6706000 7131000 7318000 3VEDFLER 22.32 5949000 6223000 5930000 6782000 6722000 6

14 >gi|7245976|pdb|1QPB|A Chain A 49741200 49088900 52205200 53847300 51928400 4AQYNEIQGWDHLSLLPTFGAK 38.45 731200 721900 793200 825300 787400 5IYEVEGMR 20.75 25440000 25120000 26570000 28350000 27210000 4NATFPGVQMK 23.45 17950000 17460000 18720000 18480000 17970000 2TPANAAVPASTPLK 20.14 4438000 4620000 4867000 4894000 4715000 4TTYVTQRPVYLGLPANLVDLNVPAK 37.03 1182000 1167000 1255000 1298000 1246000 4

15 >gi|10383781|ref|NP_009938.2| 3-phosphoglycerate kinase 87508000 87589000 92161000 94515000 96300000 4ALLDEVVK 25.23 30600000 30680000 32700000 33200000 33510000 4ELPGVAFLSEK 31.58 21140000 20890000 22170000 22420000 22650000 3IVAALPTIK 26.07 29200000 29780000 30480000 31790000 33240000 5TIVWNGPPGVFEFEK 35.57 2294000 2237000 2447000 2484000 2479000 4VLENTEIGDSIFDK 29.5 4274000 4002000 4364000 4621000 4421000 5

16 >gi|6319546|ref|NP_009628.1| Hsp26p [Saccharomyces cerevisiae] 73538400 78401200 80520000 82302800 79730600 4ADYANGVLTLTVPK 30.12 2012000 2112000 2211000 2218000 2067000 4LLGEGGLR 19.76 62940000 67250000 69090000 70820000 68710000 4NQILVSGEIPSTLNEESK 28.24 331900 350100 367100 379000 339000 5SVAVPVDILDHDNNYELK 31.72 210500 202100 207900 199800 195600 3VITLPDYPGVDADNIK 30.36 8044000 8487000 8644000 8686000 8419000 3

17 >gi|6320936|ref|NP_011015.1| Cobalamin-independent methionine synthase 6687200 6896400 7375600 7212200 7131500 4ALDADVVSIEFSK 30.14 264600 283500 288700 285000 297400 4DDANYIAEFK 25.66 672600 688900 720900 722200 737100 3VIQVDEPALR 23.5 5750000 5924000 6366000 6205000 6097000 4

18 >gi|6324212|ref|NP_014282.1| Malate synthase 5035000 5037000 5105000 5510000 5301000 4NQIDFDTPR 21.12 1721000 1658000 1749000 1825000 1776000 3YFLPEIR 29.19 3314000 3379000 3356000 3685000 3525000 4

19 >gi|6322815|ref|NP_012889.1| UDP-glucose pyrophosphorylase (UGPase) 2821500 2868400 3103300 3076200 3030600 4EGNTFLDLSVR 29.8 545900 542700 586800 581300 554200 3GGTLISYDGQVR 23.37 734600 778700 838500 829900 796400 5LLEVAQVPK 23.39 1541000 1547000 1678000 1665000 1680000 4

20 >gi|6323335|ref|NP_013407.1| Aco1p [Saccharomyces cerevisiae] 5346700 5423000 5551700 5779900 5690400 3QNVETLDIVR 24.66 765800 763500 812000 811500 783800 3SMIEYLEATGR 29.69 732000 792100 812100 776500 794800 3TIFTVTPGSEQIR 27 3578000 3591000 3640000 3919000 3826000 4WVVIGDENFGEGSSR 29.86 270900 276400 287600 272900 285800 2

21 >gi|6322323|ref|NP_012397.1| Tif2p [Saccharomyces cerevisiae] 7844800 7847300 8257700 8384100 8290900 3APQALMLAPTR 24.26 1528000 1437000 1547000 1570000 1584000 3ELALQIQK 22.62 1143000 1172000 1234000 1246000 1231000 3FDDMELDENLLR 31.75 526800 497300 519700 540100 513900 3ILISTDLLAR 30.97 3418000 3446000 3628000 3645000 3637000 3TGTFSIAALQR 27.88 1229000 1295000 1329000 1383000 1325000 4

Table 2.Analyticalprecisionof 40targetedyeastproteinsanalyzedusing aniSRMworkflow

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21 >gi|6322323|ref|NP_012397.1| Tif2p [Saccharomyces cerevisiae] 7844800 7847300 8257700 8384100 8290900 3APQALMLAPTR 24.26 1528000 1437000 1547000 1570000 1584000 3ELALQIQK 22.62 1143000 1172000 1234000 1246000 1231000 3FDDMELDENLLR 31.75 526800 497300 519700 540100 513900 3ILISTDLLAR 30.97 3418000 3446000 3628000 3645000 3637000 3TGTFSIAALQR 27.88 1229000 1295000 1329000 1383000 1325000 4

22 >gi|6321772|ref|NP_011848.1| Protein component of the small (40S) ribosomal subunit; 2970000 2874000 3055000 3127000 3061000 3QLENVSSNIVK 21.32 1064000 1017000 1051000 1075000 1071000 2YIDLEAPVQIVK 31.32 1906000 1857000 2004000 2052000 1990000 4

23 >gi|6324291|ref|NP_014361.1| Idh1p [Saccharomyces cerevisiae] 3346100 3356500 3573200 3608300 3549800 4DYAVFEPGSR 23.23 1609000 1617000 1744000 1730000 1694000 3FTVTLIPGDGVGK 29.97 633200 626600 663900 699700 657800 4IPDIDLIVIR 35.09 127500 116900 131300 127600 128000 4NIITEIGQK 24.66 976400 996000 1034000 1051000 1070000 3

24 >gi|6324950|ref|NP_015019.1| Ald4p [Saccharomyces cerevisiae] 1615700 1549900 1655100 1716800 1653700 4AFSNGSWNGIDPIDR 29.11 359400 346900 353600 398000 353600 5GDVDLVINYLK 33.18 443900 417400 456600 464400 451500 4IAPALVTGNTVVLK 27.99 812400 785600 844900 854400 848600 3IVGEAITNHPK 15.94 332700 359500 344400 358800 3SPNIVFADAELK 28.63 79770 80520 88070 92670 89610 6

25 >gi|6322066|ref|NP_012141.1| Component of the mitochondrial alpha-ketoglutarate dehydrogenase complex which catalyzes a key step in the tricarboxylic acid (TCA) cycle2714200 2740210 2881210 2860400 2927000 3

FGLEGLESVVPGIK 36.52 648600 619500 690400 665200 694700 4FLQLANEDPR 24.14 1415000 1456000 1533000 1495000 1517000 3IEQLHPFPFAQLR 33.51 103500 96210 99710 114300 106700 6LNVLSNVVR 26.62 547100 568500 558100 585900 608600 4

26 >gi|6319972|ref|NP_010052.1| Cytoplasmic ATPase 18750700 18956100 20023700 20676000 20246200 4DAGAISGLNVLR 28.04 7653000 7627000 8083000 8405000 8278000 4IINEPTAAAIAYGLGAGK 31.58 986700 953100 988700 1014000 968200 2SSNITISNAVGR 20.67 2219000 2189000 2364000 2454000 2373000 4STLEPVEQVLK 27.08 3574000 3610000 3797000 3942000 3866000 4TGLDISDDAR 19.53 4318000 4577000 4791000 4861000 4761000 4

27 >gi|6321968|ref|NP_012044.1| Eno2p [Saccharomyces cerevisiae] 5009000 4856000 5095000 5252000 5052000 3AVDDFLLSLDGTANK 34.29 3598000 3475000 3699000 3784000 3732000 3NVPLYQHLADLSK 29.66 1411000 1381000 1396000 1468000 1320000 3

28 >gi|3793|emb|CAA27203.1| unnamed protein product [Saccharomyces cerevisiae] 13948200 14400600 15149400 15513200 14744700 4ESGDFLAIDLGGTNLR 33.88 176100 169000 179700 183900 176700 3IFTVPTETLQAVTK 31.32 1171000 1077000 1164000 1276000 1174000 5INEGILQR 20.07 11010000 11510000 12070000 12270000 11660000 4IVPAEDGSGAGAAVIAALAQK 32.92 279100 247600 277700 287300 272000 5LSELIGAR 19.8 1312000 1397000 1458000 1496000 1462000 5

29 >gi|6325253|ref|NP_015321.1| Lsp1p [Saccharomyces cerevisiae] 6177700 6170600 6301900 6552900 5946100 4APTAAELQAPPPPPSSTK 21.17 360300 331000 343300 372200 356800 4ASIASSFR 18.4 2135000 2057000 2173000 2194000 2121000 2FALIAGYGK 26.92 647400 665600 654600 707700 731300 5NAAGNFGPELAR 22.2 1245000 1298000 1328000 1380000 1382000 4NIEASVQPSR 15.5 1790000 1819000 1803000 1899000 1355000 12

30 >gi|6324328|ref|NP_014398.1| Cit1p [Saccharomyces cerevisiae] 15780000 15320000 16357000 16786000 16047000 3AIGVLPQLIIDR 34.95 5418000 5130000 5593000 5678000 5521000 3ALSADLAAR 18.5 5108000 4999000 5162000 5399000 5083000 3GLVWEGSVLDPEEGIR 34.14 1159000 1060000 1158000 1200000 1144000 4LVSTIYEVAPGVLTK 31.17 1384000 1304000 1420000 1412000 1374000 3YLWDTLNAGR 30.12 2711000 2827000 3024000 3097000 2925000 5

31 >gi|6323288|ref|NP_013360.1| Hsp60p [Saccharomyces cerevisiae] 2538850 2675140 2765260 2821440 2752390 4DRYDDALNATR 18.5 91350 76440 80860 76940 80190 7LIDEYGDDFAK 24.38 1044000 1084000 1128000 1141000 1123000 3LSGGVAVIR 19.36 1164000 1267000 1301000 1333000 1305000 5TLEDELEVTEGMR 28.87 239500 247700 255400 270500 244200 4

32 >gi|487960|gb|AAB68391.1| Yhr064cp [Saccharomyces cerevisiae] 795820 788730 840400 824140 837350 3LAAEDYIGSAVK 25.38 283900 288100 308500 298600 305700 3NASNNPNELAASGAALQAR 22.95 95620 88530 103500 96140 97350 5NDVDVIANPDGER 20.51 416300 412100 428400 429400 434300 2

33 >gi|6324343|ref|NP_014413.1| Acc1p [Saccharomyces cerevisiae] 2245290 2271640 2389830 2444670 2351500 4AFLASEEAR 18.14 984200 999000 1062000 1087000 1024000 4LGGIPLGVIGVETR 32.87 704600 677500 698100 742700 703200 3SVGIGAYLVR 28.35 349900 375700 399100 408500 398600 5TGLVSVDDDIYQK 25.79 166300 181300 195700 166800 188500 6YYTFNGPNYNENETIR 26.54 40290 38140 34930 39670 37200 5

34 >gi|6320710|ref|NP_010790.1| S-adenosylmethionine synthetase 10019400 10647000 10909400 11053300 11065500 4IIVDAYGGASSVGGGAFSGK 27.34 109600 116200 120800 125400 126200 5NFDLRPGVLVK 28.36 104800 110800 118600 117900 119300 5YFIQPSGR 19.5 9805000 10420000 10670000 10810000 10820000 4

35 >gi|6323387|ref|NP_013459.1| Acetohydroxyacid reductoisomerase 5813200 5847900 6272300 6221500 6150200 4GALDWYPIFK 36.48 591600 571600 638100 611300 633500 4GINSSYAVWNDVTGK 29.74 456600 458300 485200 473200 470700 2NLFTVEDAIK 29.97 2431000 2516000 2599000 2643000 2667000 3SLEFNSQPDYR 22.93 2334000 2302000 2550000 2494000 2379000 4

36 >gi|6321013|ref|NP_011092.1| Pab1p [Saccharomyces cerevisiae] 702100 715300 755000 758400 709400 3SQLAQQIQAR 17.85 702100 715300 755000 758400 709400 3

37 >gi|3724|emb|CAA24608.1| unnamed protein product [Saccharomyces cerevisiae] 146943000 144584000 150781000 151705000 151540000 2LVSWYDNEYGYSTR 28.09 3826000 3842000 3978000 4117000 3899000 3TASGNIIPSSTGAAK 18.64 86870000 84210000 86950000 86400000 88330000 2VAINGFGR 20.4 50290000 50700000 53760000 55080000 53470000 4VVDLVEHVAK 21.45 4533000 4389000 4563000 4652000 4449000 2VVDLVEHVAKA 23.49 1424000 1443000 1530000 1456000 1392000 3

38 >gi|6321679|ref|NP_011756.1| Alpha subunit of heterooctameric phosphofructokinase 2050400 2036900 2181700 2284700 2180500 5IGDILSGR 21.04 1787000 1774000 1907000 2007000 1908000 5WLATLQGVDAVK 29.86 263400 262900 274700 277700 272500 2

39 >gi|24158771|pdb|1KB9|A Chain A 2409500 2404000 2597400 2667200 2597400 5DFQSYIVSSLPGSTDK 32.35 287800 279400 299600 296300 276400 3DSGLWGFSTATR 29.49 824400 792700 846800 894200 883800 4EGLALSSNISR 22.78 1111000 1148000 1259000 1271000 1239000 5SLDFLNQSFIQQK 33.02 186300 183900 192000 205700 198200 4

40 >gi|114589|sp|P05626|ATPF_YEAST ATP synthase subunit 4 2850000 2822100 2860200 2982000 2975800 3ANSIINAIPGNNILTK 31.82 213600 218400 225000 226400 218100 2AVLDSWVR 26.39 1263000 1235000 1258000 1350000 1334000 4IDSVSQLQNVAETTK 25.63 149400 144700 161200 145600 149700 4VSDVLNASR 15.31 1224000 1224000 1216000 1260000 1274000 2

Peak Area Peak Area Peak Area Peak Area Peak Area# Protein Peptide RT n=1 n=2 n=3 n=4 n=5 CV%

11

ConclusionsThe newly developed intelligent SRM (iSRM) technologyprovides the easiest and fastest way to develop the robust,accurate, and sensitive SRM assays for targeted proteinquantification. A validated, highly complex iSRM assay tosimultaneously quantify and identify 40 yeast proteins wasdeveloped in few hours by using Pinpoint software andthe iSRM workflow.

References1. Gerber, S.A., Rush, J., Stemman, O., Kirschner, M.W & Gygi. S.P.

Absolute quantification of proteins and phosphoproteins from cell lysatesby tandem MS. Proc. Natl. Acad. Sci. USA 100, 6940-6945 (2003).

2. Anderson, L. & Hunter, C. L. Quantitative mass spectrometric multiplereaction monitoring assays for major plasma proteins. Mol. Cell.Proteomics 5, 573-588 (2006).

3. Kiyonami, R., Peterman, S. M., Viner, R., Prakash, A. & Zabrouskov, V. anew methodology for targeted peptide quantification in complex mixturesusing a high resolution triple quadrupole mass spectrometer. Applicationnote of Thermo Fisher Scientific, #412.

4. Kiyonami, R., Schoen, A., Prakash, A., Nguyen, H., Peterman, S. M.,Selevsek, N., Zabrouskov, V., Huhmer, A. & Domon, B. IncreasedQuantitative Throughput and Selectivity for Triple Quadrupole MassSpectrometer-Based Assays Using Intelligent SRM (iSRM). ASMS 2009poster, #W313.

5. Prakash, A., Kiyonami, R., Schoen, A., Nguyen, H., Peterman, S. M.,Huhmer, A., Lopez, M. & Domon, B. Integrated workflow to designmethods and analyze data in large-to-extremely-large scale SRMexperiments. ASMS 2009 poster, #TH695.

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