Coverage of Low Abundance Plasma Proteins - Ahn et al. - 2021 - Unknown

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pubs.acs.org/jprArticleUseofaRecombinantBiomarkerProteinDDALibraryIncreasesDIACoverageofLowAbundancePlasmaProteinsSeongBeomAhn,*KarthikS.Kamath,AbidaliMohamedali,ZainabNoor,JemmaX.Wu,DanaPascovici,SubashAdhikari,HarishR.Cheruku,GillesJ.Guillemin,MatthewJ.McKay,EdouardC.Nice,andMarkS.Baker*CiteThis:J.ProteomeRes.2021,20,2374−2389ReadOnlineACCESSMetrics&MoreArticleRecommendations*sıSupportingInformationABSTRACT:Credibledetectionandquantificationoflowabundanceproteinsfromhumanbloodplasmaisamajorchallengeinprecisionmedicinebiomarkerdiscoverywhenusingmassspectrometry(MS).Inthisproof-of-conceptstudy,weemployedamixtureofselectedrecombinantproteinsinDDAlibrariestosubsequentlyidentify(notquantify)cancer-associatedlowabundanceplasmaproteinsusingSWATH/DIA.TheexemplarDDArecombinantproteinspectrallibrary(rPSL)wasderivedfromtrypticdigestionof36recombinanthumanproteinsthathadbeenpreviouslyimplicatedaspossiblecancerbiomarkersfrombothourownandotherstudies.TherPSLwasthenusedtoidentifyproteinsfromnondepletedcolorectalcancer(CRC)EDTAplasmasbySWATH-MS.Most(32/36)oftheproteinsusedintherPSLwerereliablyidentifiedfromCRCplasmasamples,including8proteins(i.e.,BTC,CXCL10,IL1B,IL6,ITGB6,TGFα,TNF,TP53)notpreviouslydetectedusinghigh-stringencyproteininferenceMSaccordingtoPeptideAtlas.TherPSLSWATH-MSprotocolwascomparedtoDDA-MSusingMARS-depletedandpostdigestionpeptidefractionatedplasmas(herereferredtoasahumanplasmaDDAlibrary).Ofthe32proteinsidentifiedusingrPSLSWATH,only12couldbeidentifiedusingDDA-MS.The20additionalproteinsexclusivelyidentifiedusingtherPSLSWATHapproachwerealmostexclusivelylowerabundance(i.e.,<10ng/mL)proteins.TomitigatejustifiedFDRconcerns,andtoreplicateamoretypicallibrarycreationapproach,theDDArPSLlibrarywasmergedwithahumanplasmaDDAlibraryandSWATHidentificationrepeatedusingsuchamergedlibrary.Themajority(33/36)ofthelowabundanceplasmaproteinsaddedfromtherPSLwerestillabletobeidentifiedusingsuchamergedlibrarywhenhigh-stringencyHPPGuidelinesv3.0proteininferencecriteriawereappliedtoourdataset.TheMSdatasethasbeendepositedtoProteomeXchangeConsortiumviathePRIDEpartnerrepository(PXD022361).KEYWORDS:recombinantproteinspectralDDAlibrary(rPSL),SWATH,lowabundanceplasmaproteinidentification,DownloadedviaUNIVOFCONNECTICUTonMay16,2021at08:33:02(UTC).cancerbiomarkersSeehttps://pubs.acs.org/sharingguidelinesforoptionsonhowtolegitimatelysharepublishedarticles.■INTRODUCTIONconcentrationof∼50mg/mL,whereasthecytokinesIL-6or2Biologicalfluidslikeplasma,serum,saliva,andurineareIL-8arepresentatthelowpg/mLrange.Themaskingoflowcommonlyusedforclinicaldiagnosticapplications.Althoughabundanceproteins(LAPs)bythesehighlyabundantproteinsplasmaishighlyheterogeneousacrossawiderangeofprotein(HAPs)makesitdifficulttodetect,identify,andquantifymanyconcentrationscomparedtomanyotherbiospecimens,itisa3particularlyattractivesourceforidentificationofdisease-disease-specificproteinbiomarkerswithouteitherextensiverelatedproteinbiosignatures.1Plasmacollectionisminimally45fractionationorspecificenrichment(e.g.,glycoproteins,invasiveandhencereadilyavailable.Itperfusesalltissuesand6phosphoproteins).hasarelativelyconstantvolume(∼5Linanadult)andthereforehasbeensuggestedtorepresentpatho-physiochem-icalsnapshotofanindividualatanygiventime.Received:November7,2020However,theanalysisofplasmaproteinsbytandemmassPublished:March22,2021spectrometry(MS)isanalyticallychallengingbecauseofthepresenceofmany“housekeeping”liver-derivedproteinscoveringalargedynamicproteinconcentrationrange(>12orders).Forexample,humanserumalbuminhasatypical©2021AmericanChemicalSocietyhttps://doi.org/10.1021/acs.jproteome.0c008982374J.ProteomeRes.2021,20,2374−2389

1JournalofProteomeResearchpubs.acs.org/jprArticleTable1.Cancer-AssociatedProteins(36Proteins)UsedtoGeneratetherPSLDDASpectralLibraryfoundinplasmabbyMScancersahumanplasmaconcentrationgenenameproteinname(pg/mLtoμg/mL)Y/Nrefscancersrefs14,327ADAMDEC1Adisintegrinandmetalloproteinasedomain-like76ng/mLYColorectalproteindecysin-1c3334BTCProbetacellulin4ng/mLNOvarian14,32,35,3637C1QCComplementC1qsubcomponentsubunitC912ng/mLYProstate1438−40CEACAM5Carcinoembryonicantigen-relatedcelladhesion1ng/mLYColorectal,Lung,molecule5Gastric14,41,4243CPQCarboxypeptidaseQ29μg/mLYLiver14,44,4546CST3Cystatin-C5μg/mLYHeadandNeckd4748−50CXCL8(IL8)C-X-Cmotifchemokine8(Interleukin-8)14pg/mLNColorectal,Brain,Breastd4751CXCL10(IP10)C-X-Cmotifchemokine10(Interferongamma-724pg/mLNBreastinducedprotein10)41,5253,54CXCL12(SDF-C-X-Cmotifchemokine12(stromalcell-derived1ng/mLYHeadandneck,1α)factor1)Esophageal14,4255EGFPro-epidermalgrowthfactor2ng/mLYLung14,5657EGFREpidermalgrowthfactorreceptor12ng/mLYBreastd4758IL1BInterleukin-1beta1ng/mLNOrald4759,60IL6Interleukin-61ng/mLNColorectal,Prostate41,5261ITGAVIntegrinalphaV2ng/mLYOvarian14,4162ITGB1Integrinbeta12μg/mLYBreastc3361,63ITGB6Integrinbeta62ng/mLNColorectal36,4164KLK3Plasmakallikrein26ng/mLYProstate5265MIAMelanoma-derivedgrowthregulatoryprotein9ng/mLYMelanoma52,56,6667,68MMP2Matrixmetalloproteinase2812ng/mLYColorectal,Breast14,4169MMP3Matrixmetalloproteinase-3/Stromelysin-179ng/mLYOvarian14,41,5667,68MMP9Matrixmetalloproteinase9190ng/mLYColorectal,Breast1670MUC1Mucin-1−YBreast14,4171PDGFBPlatelet-derivedgrowthfactorsubunitB323pg/mLYColorectal41,4272PFN1Profilin-1129ng/mLYLung1460PLAUUrokinase-typeplasminogenactivator794pg/mLYProstate1473−75PLAURUrokinaseplasminogenactivatorsurfacereceptor2ng/mLYColorectal,Lung,Prostate14,36,41,4276PLGPlasminogen302μg/mLYOvarian14,41,4277PTENPhosphatasetensinhomologue295pg/mLYProstate41,4278S100A8ProteinS100-A8269ng/mLYBladder41,4278S100A9ProteinS100-A92μg/mLYBladderc3340TGFAPro-transforminggrowthfactorα15pg/mLNGastric35,41,4479,80TIMP1Metalloproteinaseinhibitor1269ng/mLYColorectal,Breast14,32,5281TIMP2Metalloproteinaseinhibitor2151ng/mLYColorectale8283TNFTumournecrosisfactor2ng/mLNBreast1484TNFRSF1ATumournecrosisfactorreceptorsuperfamily5ng/mLYProstatemember1Ad4785TP53Cellulartumorantigenp53398pg/mLNBladdera86bHumanplasmaproteomedatabaseandPeptideAtlasPlasmaBuild2017(http://www.peptideatlas.org).BasedonPeptideAtlasPlasmaBuildcde2017(http://www.peptideatlas.org).Observedincolorectalcancertissue.Observedinbreasttissue.Observedinimmunecells.Oneapproachtobroadenthe“reach”ofplasmaproteomicsandfractionationmethodsindependentlyhastypicallybeenwhilemitigatingmaskingeffectsinvolvesremovalofHAPslimitedtosmallpilotdiscoverystudies3andisnotyetusingimmunodepletion,sincethemajor14or20HAPsamenabletoautomatedhigh-throughputlargeclinicalstudies.represent∼90and∼97%ofthetotalplasmaproteomeWhenshotgunproteomicsisusedtoidentifydisease-related7content,respectively.Anotherapproachistouseextensivebiomarkers,10complexproteinmixturesareroutinelyenzy-multidimensionalpeptidefractionationafterplasmaproteolytic8,9maticallydigestedorchemicallycleaved,withsubsequentdigestionthatfacilitatestheanalysisofpeptidesfromLAPs.peptidesseparatedbyHPLCfollowedbyidentificationusingBothmethodsarethoughttoallowtheproteometobetandemMS/MS.Utilizingshotgunproteomicsincombinationexploredingreaterdepththatbetterreflectspathophysiologyorrevealproteinsthatmaybedisease-ordiseasestage-withdepletionorfractionationcanallowtheinference,specific.7,10Moreover,depletedand/orfractionatedplasmaidentificationandquantitationof≥1000plasmaproteins14,15samplesareamenabletoanalysisbyantibody-basedfromasinglestudy.Despitethehighnumberofprotein11,1213technologiesand/orMS.However,theuseofdepletionidentificationsusingshotgunproteomics,disease-specific2375https://doi.org/10.1021/acs.jproteome.0c00898J.ProteomeRes.2021,20,2374−2389

2JournalofProteomeResearchpubs.acs.org/jprArticleFigure1.Experimentalworkflow.(a)Constructionofrecombinantproteinspectrallibrary.Atotalof36cancer-associatedbiomarkerswereselectedfromtheliteratureandourownstudies(Table1)andsortedinto4groups(A−D)basedontheirmolecularweight(MW).Eachgroupof9proteinswasspikedwithvitronectinforretentiontime(RT)alignment.Proteinswerereduced,alkylated,anddigestedwithtrypsin.DDAwasusedforproteinidentificationusingaSCIEXTripleTOF6600.Datasetswereconcatenatedtogeneratearecombinantproteinspectrallibrary.(b)Constructionofhumanplasmaproteinspectrallibrary.CRCplasma(80fromstageI−IVCRCs)and20healthyplasmasampleswerepooledanddepletedthetop14highabundanceproteinswithanAgilentMARS-14depletioncolumn.ThedepletedsamplesweredigestedwithtrypsinfollowedbypeptidefractionationusinghighpHreverse-phasedHPLC.ADDAmethodwasemployedasin(a)toconstructtheplasmaproteinDDAspectrallibrary.(c)SWATH/DIAproteinidentificationusingrPSLormergedlibraries.TheconstructedrPSLormergedlibraries(rPSL+plasmaproteinspectrallibrary)werecombinedwithSWATH/DIAMSanalysistodeterminewhetheritwaspossibletodetecttrypticpeptidespectraofthe36cancer-associatedproteinsinnondepletedhumanplasmasamplesobtainedfromCRCpatients(n=5).Followingsamplepreparation(reduction,alkylation,andtrypticdigestion),SWATH/DIAMSanalysiswasperformedforpeptide/proteinidentification.PeakViewandSkylinewereemployedforMSdataextractionandpeakselectionwith1%FDRfiltering.Identifiedproteinswerefurtherfilteredusinghighstringencyproteinidentificationcriteria(HPPguidelinev3.0).regulatoryproteinsexpressedatextremelylowlevelsfrequentlyrecordofallindividualpeptidesrepresentedinaconvoluted,remainmaskedbyHAPs.buthighlystructuredmanner.Usingastandardanalyticalpipeline,HUPO’sBiology/However,toachieveaccuratequantificationusingSWATH-Disease-HumanProteomeProjectHPP(B/D-HPP)plasmaMS,itiscrucialtohavepriorknowledgeofthePSM(peptideproteometeamhasanalyzed178individualexperiments.Theyspectrummatches)andchromatographicbehaviorofallhaverecentlyreportedatotalof3509plasmaproteinspeptidesofinterest.ThesePSMscanbeusedtoextractidentifiedwiththeimpositionofcommunity-endorsedhigh-peptide-specificinformationfromobservedMSspectraldata17stringencyproteininferenceparameters.16ThisHPPteamalsousingPQPs(peptidequeryparameters).Thisinformationincludespeptidesequence,m/zvaluesofthedominantobtainedevidenceforanadditional1300proteins,although16precursorionofthepeptide,chargestate,fourtosixmostthiswasatlowerstringency,lesscredibleevidence,intensefragmentionm/zvaluesofpeptide(s)underillustratingthedifficultiesassociatedwithdeepplasmafragmentationconditions,informationaboutanticipatedproteomeanalyses.fragmentation,andexpectedLCretentiontimes.Asidefromestablishedshotgunapproaches,emergingMSThesePQPsarecommonlyobtainedfromspectraldatatechnologiesmayhavethepotentialtoovercomesomeknownacquiredfromDDA(data-dependentacquisition)runslimitationsinproteinidentificationandquantification.One17performedpriortoaDIAexperiment.Thedatasetssuchtechnologyissequentialwindowacquisitionofallemployedarecommonlyreferredtoas“peptidespectraltheoreticalmassspectra(SWATHMS),adata-independentlibraries”.GeneratingpeptidespectrallibrariesusingDDAisacquisition(DIA)methodthatallowsdeepproteomecoveragetime-consuming,complicatedandcurrentlyamajorlimitationwiththepromiseofcomprehensive,accurateandreproducibleofDIAMS,includingSWATH.18,19Spectrallibrariesare17quantitation.InSWATH-MS,allionizedpeptidesintheusuallyequally,ifnotmore,complexthanthesamplebeingsamplethatfallwithinaspecifiedmassrangearefragmentedinanalyzed(althoughnotaprerequirement)andthequalityofasystematicandunbiasedfashionusingprecursorisolationthedatadependsonfactorssuchasisolationwindowwidths,1720windows.Theresultantdatasetconstitutesacompletefragmentionresolutions,dwelltimesandcycletimes.Itis2376https://doi.org/10.1021/acs.jproteome.0c00898J.ProteomeRes.2021,20,2374−2389

3JournalofProteomeResearchpubs.acs.org/jprArticle7,29crucialtogeneratespectrallibrariesusinganidenticalemployedpreviously.Experimentaldesigndetailstoinstrumenttypewithidenticalsettingswhenperformingbothspecificallyinvestigatethequestionofwhetherlowabundant19DDAandDIApartsofallrelatedexperiments.Additionally,proteinscouldbeidentifiedfromnondepletedplasmaaretominimizevariation,anumberofinformaticstoolshavebeenprovidedinFigure1.Proteinquantitationwasnotinvestigated21developedandimplemented.DIAspecificsoftware(e.g.,asthiswasbeyondthescopeofthispresentproof-of-principlePeakView,Skyline)isthenfrequentlyusedtoidentifyuniquetechnicalstudy.peakgroupsassociatedwithpreviouslygeneratedDDATodemonstratetheabilityofarPSLSWATHtoallowspectrallibrariesthatcanthenbeusedtoaccuratelyidentifydetectionoflowabundancecancer-associatedproteinsfromandquantifytargetpeptidesobservedintheDIApartofannondepletedplasma,wecomparedproteinidentificationsfrom19rPSLSWATHtopreviousplasmaproteinidentificationexperiment.GiventhatSWATH/DIAisoftenreliantonpreviouslymethods(i.e.,DDAshotgunproteomicsusingMARS-14-generatedDDApeptidespectrallibraries(asopposedtodepletedorpeptidefractionatedtrypticallydigestedCRClibrary-freeapproaches),manystudieshaveattemptedtoplasmaproteins,referredtoasthe“plasmaproteinDDAincreasethedepthofDDAlibraries.Somehaveusedarangeofspectrallibrary”).protein/peptidefractionationstrategies(e.g.,HAPdepletionTomitigateFDRconcernsduetoanyrelianceonjusttheand/orcombinationsofpeptidefractionationmethods)priorsmall36recombinantproteinrPSL,ourrPSLlibrarywasalso7,22mergedwithamuchlargerhumanplasmaproteinDDAtoDDAexperiments.OtherresearchershavegeneratedlargerlibrariesbycombiningtwoormoreexistingDDAspectrallibrary(containingdataidentifying742plasma23proteinsathigh-stringencyHPPGuidelinesv3.0criteriaoflibraries.Equally,softwaretoolshavebeendevelopedthat18,24≥2peptidesnon-nested,uniquelymappingpeptidesof≥9facilitatelibraryconcatenation.Clearly,generationofcomprehensive,high-qualitypeptidespectrallibrariesforaminoacidslengthandlimitedto≤1missedcleavage)and30,31high-qualitySWATH/DIAanalysisiscrucial,astheseSWATH-MSproteinidentificationrepeated.representthebiological/proteomespaceofbiospecimensOurapproachindicatesthattheuseofarPSLSWATHbeinginterrogated.libraryfacilitatesidentificationofmanyverylowabundanceUnfortunately,becauseofarequirementforlibrariestobe(somepreviouslyundetectable)plasmaproteinsspecificallyinbasedonpriorDDAexperiments,mostSWATH/DIAplasmathelowabundancepg−ng/mLrange.rPSLSWATHstudiesareunabletoidentifyLAPs.Rather,mostchoosetoapproaches,therefore,appeartohavethepotentialtoallowfocusonproteinquantitationofonlypreidentifiedlibrarymuchdeeperplasmadiscoveryintolowabundanceplasmaproteinsfoundfromcombinedsamplesbyDDA.Althoughproteinsthatpotentiallyhaveutilityasdiagnostic,prognostic,newtoolslikelibrary-freeDIAapproaches25continuetoand/ortheranosticindicatorsforcancerorforthedetection/evolve,library-basedapproachesremainwidelyusedacrosssurveillanceofotherdiseases.SWATH/DIAapplications.Thisstudyfocusesonimprovingplasmaproteincoverageusinganovellibrary-basedstrategy.■MATERIALSANDMETHODSHere,weemployanewapproachtodetermineifitisEthicsStatementandPlasmaSampleCollectionpossibletoidentifylowerabundancecancer-associatedplasmaproteinsusingSWATH/DIA.ADDAspectralpeptidelibraryThisstudywasperformedwithapproval#5201200702fromderivedfromtrypticpeptidesfromacarefullychosensetof36MacquarieUniversityHumanResearchEthicsCommittee.humanrecombinantproteinswasconstructed(Table1).EachTheEDTA-plasmacohortcontained100individualspatientsofthebiomarkerproteinsselectedhadbeenpreviously(80clinicallystagedCRCpatientsand20healthycontrols)implicatedinhumancancerandmightbeexpectedtobeprocuredfromtheVictorianCancerBiobank,Melbourne,present(albeitatlowconcentrations)inhumanCRCplasma.Australia.Sampledetailsandpreparationmethodshavebeen7,29Thepreviouslyreportedplasmaconcentrationsmeasuredbydescribedpreviously.anytechnologyofthoseselected36proteinsaresummarizedRecombinantProteinsinTable1.ThissetwasbaseduponcancerbiomarkerproteinRecombinantproteinsADEMDEC1(TP721090),BTCcandidatespreviouslyidentifiedintheliteratureandinclude(TP723036),C1QC(TP761200),CPQ(TP760108),BTC,C1QC,CEACAM5,CPQ,CXCL8,CXCL10,CXCL12,CXCL10(IP10,TP723726),CXCL8(IL8,TP721122),EGF,EGFR,IL1B,IL6,ITGAV,ITGB1,KLK3,MIA,MMP2/MMP2(TP723320),MUC1(TP760771),PDGFB3/9,MUC1,PDGFB,PFN1,PLAU,PTEN,S100A8/9,(TP723355),TGFA(TP723858),TIMP2(TP723886)andTGFA,TIMP1/2,TNF,TNFRSF1A,andTP53.Inaddition,TNFRSF1A(TP723870)werepurchasedfromOriGeneourpriorultradepletedplasmacolorectalcancerstudieshaveTechnologies,CEACAM5(4128-CM),CST3(1196-PI),implicatedADAMDEC1,CST3,ITGB6,andPLAURasCXCL12(350-NS),EGFR(1095-ER),IL1B(201-LB),IL6potentialearlyclinicalstageCRCbiomarkersandthesehave(206-IL),KLK3(1344-SE),MIA(9250-IA),MMP3(513-beencombinedwiththeliteraturebiomarkercandidatesabove7,26−29MP),MMP9(911-MP),PLAU(1310-SE),PLAUR(807-tocreateaninitialtailoredrPSL.Importantly,9oftheUK),PTEN(847-PN),S100A8/A9(8226-S8),TIMP1(970-recombinantproteinsusedtoconstructthisrPSLhavenotTM),TNF(210-TA),TP53(SP-454)andVN(2308-VN)beenpreviouslyidentifiedinhumanplasmabyMS,accordingfromR&DSystems,PFN1(NBP1-30215),PLG(H00005340-tothelatestplasmabuildfromPeptideAtlas(Table1).TheP01),ITGAV(H00003685-P01),ITGB1(H00003688-P01)constructedrecombinantproteinspectrallibrary(rPSL)wasandITGB6(H00003694-P01)fromNovusBiologicalsandcoupledtoaSWATH-MSanalysis(rPSLSWATH)workflowEGF(MBS650012)fromMyBioSource.todetermineifitwaspossibletoobservehigh-stringencytrypticpeptidespectraforanyofthese36cancer-associatedSamplePreparationproteinsinnondepleted,clinicallystagedCRCpatientplasmas.ForrPSLconstruction,selectedrecombinantproteinswereTheplasmasusedinthisstudyareidenticalwiththosepooled(Table1)intofourgroupsbasedonsimilarmolecular2377https://doi.org/10.1021/acs.jproteome.0c00898J.ProteomeRes.2021,20,2374−2389

4JournalofProteomeResearchpubs.acs.org/jprArticleweight(MW)(namely,groupsA:5−16kDa,B:17−30kDa,Peptideuniqueness(i.e.,unitypicpeptides)wasconfirmed88C:30−55kDa,andD:70−135kDa).SampleswerereducedusingtheneXtProtuniquenesschecker.with5mMdithiothreitol(DTT)at60°Cfor30minfollowedHighStringencyPeptide/ProteinSelectionbyalkylationwith25mMiodoacetamide(IAA)atroomHPPguidelinesv3.0wereappliedfortheapplicationofhightemperaturefor30mininthedark.Sampleswerethen30stringencyproteininferences.Maximumof1missedcleavagedigestedwithsequencinggradeporcinetrypsin(Promega)atawasallowedforeachunitypicandnon-nestedpeptide,withproteasetosubstrateratioof1:30at37°Cfor16h.PeptidetrypticcleavagetowardtheC-terminalsideoftheR/KresiduesmixturesweredesaltedandcleanedwithC18OMIXtipsbutnotwhenimmediatelyfollowedbyaProlineresidue.(Agilent)accordingtothemanufacturer’sprotocol,followedGreaterthanorequalto2peptidesperproteinwasrequiredbydryingusingvacuumcentrifugation.forproteinidentification.Forthehumanplasmalibraryconstruction,all100plasmasampleswerepooledandimmunodepletedusinganAgilentSpectralLibraryMergingMARS-14highcapacityaffinitycolumnwithaAgilent1260TheindependentlygeneratedrPSLandhumanplasmaspectral7HPLCsystemasdescribedpreviously.Thedepletedplasmalibraryweremergedintoonespectrallibraryusingapreviouslysampleswerethenreduced,alkylatedanddigestedaspublishedalgorithm,SWATHXtend.18TherPSLwasusedasdescribed.Digestedpeptideswerefractionated(total20theseedlibrarywhenmerging.Nomodificationsandfractions)usingaZORBAX300Extend-C18(2.1×150miscleavedpeptideswereremoved.Onlypeptideswithmm,3.5μm)columnona1260HPLCsystem(Agilent,Santaconfidence>0.99wereconsidered.Thematchingquality7Clara,CA,USA)asdescribedpreviously.FractionedpeptidesbetweenthetwolibrariesisexcellentwiththesquaredweredesaltedandcleanedwithC18OMIXtipsanddriedbyretentiontimecorrelationof0.97,estimatedretentiontimevacuumcentrifugation.errorof1.74minandrelativeionintensitycorrelationof0.9.SpectralLibraryGeneration(DDA)Themergedspectralibrarywasoutputtedinatab-delimitedTherPSL(groupsA−Dabove)andthehumanplasmaspectralPeakViewcompatibletextformat.library(using20fractionatedpeptidesasdescribedabove)DIA/SWATH-MSweregeneratedindependently.DDAproteinidentificationwasASCIEXTripleTOF6600coupledwithEksigentUltraperformedonaSCIEXTripleTOF6600(SCIEX,Framing-nanoLCsystemwithidenticalmobilephaseconditionstoham,MA)coupledtoanEksigentUltrananoLCsystemthosedescribedabovewasusedforSWATH-MS.For(EksigentTechnologies,Dublin,CA).PeptideswereinjectedSWATH,datawasacquiredusingashorter60minLConto200μmID,3.5cm-lengthpeptide-trapcolumnspackedgradient(5−35%mobilephaseB)at600nL/min.TheeluentinhousewithaC18support(2.7μmparticlesize,HaloC18)wassubjectedtopositiveionnanoflowelectrosprayMSforpreconcentrationanddesaltedataflowrateof5μL/minanalysis.Initially,theprecursorm/zfrequenciesfromfor3minwith0.1%formicacid(v/v)and2%acetonitrile(v/previouslygeneratedplasmaproteomeDDAdatawereusedv).Afterdesalting,thepeptidetrapwasswitchedin-linewithatodeterminethem/zwindowsizes.SWATHvariablewindowcHiPLCC18column(15cm×200μm,3μm,ChromXPacquisitionwithasetof100overlappingwindowswasC18-CL,120Å,25°C,SCIEX)andpeptideswereelutedusingconstructedcoveringthemassrangem/z400−1000.Inalinear120mingradientfrom5%acetonitrileto35%mobileSWATHmode,TOF-MSsurveyscanswereacquired(m/zphaseB(B:99.9%acetonitrile,0.1%formicacid)at600nL/350−1800,0.05s)thenthe100predefinedm/zrangesweremin.InDDAmode,aTOFMSsurveyscanwasacquiredatm/sequentiallysubjectedtoMS/MSanalysis.Productionspectraz350−1500with0.25saccumulationtime,withthe20mostwereaccumulatedfor30msinthemassrangem/z200−2000intenseprecursorions(2+to5+;counts>200)inthesurveywithrollingcollisionenergyoptimizedforlowedm/zinm/zscanconsecutivelyisolatedforsubsequentproductionscans.window+10%.Dynamicexclusionwasusedwithawindowof30s.ProductDIA/SWATHPeakExtractionionspectrawereaccumulatedfor100msinthemassrangem/z100−1800withrollingcollisionenergy.DIAspectralalignmentandtargeteddataextractionwereDDAdatawereanalyzedusingProteinPilot(V5.0,SCIEX)performedindependentlyusingtwosoftwarepackages;Skyline2089withtheParagonalgorithm.TheHomosapiensproteinsoftware(Version4.1.0.18169)andPeakView(SCIEX,sequencedatabasewithreviewedentrieswasobtainedfromUSA).Inbothinstances,thesameDDAbasedspectrallibrarySwissProt(42388entriesincludingcanonicalproteinsand(∼950peptidesand∼23000transitions)wasutilizedtoisoforms,2018version).Thesearchparameterswereasexecutethetargeteddataextractions.DIAchromatogramsfollows:sampletype:identification;cysalkylation:iodoaceta-wereextractedfromSWATH-MSdatafiles(n=5permide;digestion:trypsin;instrument:TripleTOF6600;IDcondition:replicatesindicatethetechnicalinjectionrepli-focus:biologicalmodifications;precursorpeptidemasscates).tolerance:±50ppm.Areverse-decoydatabasesearchstrategyPeakExtractionUsingPeakViewwasusedwithProteinPilot,withthecalculatedproteinat1%IonlibraryandSWATHdatafileswereimportedintoFDRandadetectedproteinthreshold[UnusedProtScorePeakView2.1withSWATHquantitationplug-in(SCIEX).(Conf)]>:1.30(95.0%).ThedatahasbeendepositedtoRetentiontimesforallCRCsampleSWATHdatafileswereProteomeXchangeConsortiumviathePRIDEpartneralignedusinglinearregressionbyselecting5endogenousrepository(PXD022361).peptidesacrosstheelutionprofile.Thetop6fragmentionsforInSilicoPeptideRepertoireofRecombinantProteinseachpeptidewereextractedfromtheSWATHdatausing75RecombinantproteinsweredigestedinsilicotoidentifyppmtargetXICwidth,peptideconfidencethresholdof≥0.99,uniquelymappingnon-nestedpeptidesofminimumlengthanda10minretentiontimeextractionwindow.Afterdata87nineaminoacids,similartoourpreviousinsilicoanalysis.processing,peptideswithconfidence>99%andFDR<1%2378https://doi.org/10.1021/acs.jproteome.0c00898J.ProteomeRes.2021,20,2374−2389

5JournalofProteomeResearchpubs.acs.org/jprArticleTable2.PeptidesDetectedandProteinCoverageforEachrPSLRecombinantProteinUsedtoConstructtheExperimentalaDDASpectralLibraryandTheoreticalinSilicoAnalysisrPSLconstructionDDA-MS(experimentallyobserved)insilicoanalysisccDDA-MSdatawithdefaultHPPguidelinev3.0(≤1missedHPPguidelinev3.0(zeromissedbsettingcleavage)cleavage)coverageMWproteincoverageproteincoverageproteincoverageobserved/genename(kDa)peptides#(%)peptides#(%)peptides#(%)expectedADAMDEC152.781839103619700.52BTC19.7518325625551.13C1QC25.77617485212810.64CEACAM576.804549143225870.37CPQ51.8910082256917740.93CST315.8010362166610.26CXCL8(IL8)11.1016511122250.48CXCL10(IP10)10.8820683302271.12CXCL12(SDF-1α)10.6728743458153.00dEGF6.22171002872791.10EGFR134.285538263356770.42IL1B30.75355464510760.59IL623.7261887757481.55ITGAV116.0410264445452790.68ITGB188.422326152437760.32ITGB685.944449224634810.57KLK328.74238566712740.91MIA14.5121755497550.89MMP273.886978267228701.03MMP353.986884166416650.99MMP978.467670286226720.85MUC1122.1028125931960.09PDGFB27.28434152310550.42PFN115.0548987848811.03PLAU48.513258154822760.63PLAUR36.98225694717690.67PLG90.575856204434730.60PTEN47.174685167427661.12S100A810.8418833373460.79S100A913.2429944685681.00TGFA17.01242611312760.17TIMP123.1741756379710.52TIMP224.4033697577541.06TNF25.64165254410780.57TNFRSF1A50.50303873421720.48TP5343.655790158919701.27abTableS2containsdetailsincludingsequencesforallpeptides.Includesnestedpeptides,allpeptideswith≥7aminoacidsand≤2missedccleavage.Non-nestedunitypicpeptideswith≥9aminoacids,≤1missedcleavageforpeptidesobservedinexperimentalDDArecombinantproteindspectrallibrary(rPSL),zeromissedcleavageforinsilicoanalysis.RecombinantEGFusedinthisstudywastheactive53aminoacidformofEGF(https://www.mybiosource.com/egf-active-protein/epidermal-growth-factor/650012)withMWof∼6kDa.(basedonchromatographicfeatureafterfragmentextraction)Alistoftargetproteinswasgeneratedbyimportingtheionwereusedforquantitation.Sharedandmodifiedpeptideswerelibrarywhichwasfilteredtoremoveduplicatepeptidesandexcluded.ThesumofMS2ionpeakareasofSWATHpeptidesof<7aminoacidsinlength.Fortargetedpeptides,thequantifiedpeptidesforindividualproteinswereexportedtotop-rankedsixyandbtransitionionswithchargesupto+3calculatetheproteinpeakareas.wereselectedtogetherwiththeircorrespondingdecoy-PeakExtractionUsingSkylinetransitiongroups,generatedbyshufflingsequencesfromtheToimportSWATHdata,isolationwindowschemesrangingtargetedpeptides.Detailedpeptideandtransitionsettingsarefrom399m/zto1000m/zwereextractedfromdatafiles.ToprovidedinTableS1.Chromatogramswereextracteddirectlyperformretentiontime(RT)calibrationandtodesignaRTfromtherawdatafileswithinawindowof20min(±10min)predictor,anindexedretentiontime(iRT)calculatorusing12aroundthepredictedtimes(witharesolvingpowerof30000highintensityendogenousvitronectinandhumanserumforTripleTOF6600).Oninspectionoftheextractedpeaks,italbuminpeptideswithretentiontimesspanningthewholewasnotedthatsomeofthepeaksappearedslightlyoutsideofgradientwereused.thenarrow10minwindow.Thewindowsizewastherefore2379https://doi.org/10.1021/acs.jproteome.0c00898J.ProteomeRes.2021,20,2374−2389

6JournalofProteomeResearchpubs.acs.org/jprArticleincreasedto20mintoincludetheserefinedmanuallyThehighestpeptidenumberdetectedintherPSLwasforinspectedpeaks.integrinITGAV(102peptidesreflecting64%full-lengthForSWATH-MSdata,chromatographicpeakswereITGAVcoverage)whilethelowestwasCST3(10peptides90integratedusingmProphetpeak-scoringmodel,whichisaand36%full-lengthcoverage).Twenty-eightproteinsoutofsemisupervisedlearningalgorithm,toidentifycorrectpeaks.the36chosenhadgreaterthan40%proteincoverage.EGFPeakscoringmodelswererefinedusingfeaturescoresofhadthehighestcoverage(100%)withmucinMUC1havingpeptides,estimatingQ-valuesforeachpeak.Afalsediscoverythelowestcoverage(12%)(Table2andTableS2).rate(FDR)of1%(Q-valuecutoff0.01)wasappliedandpeaksTofacilitatedevelopmentofsubsequenttargetedassayswerefilteredbyapplyingadotproduct(dotP)cutoffof0.6(SRM,MRM,orPRM)forfuturebiomarkerstudies,wewhichshowsthecorrelationbetweenobserved(library)andundertookacomparativeanalysisonexperimentallyobservedmeasured(DIA)spectra.uniquelymapping(unitypic)peptidesagainstaunitypicPeptideandproteinintensitieswerecalculatedbymanuallypeptiderepertoirederivedinsilicofromthe36recombinantsummingaveragepeakareasofrespectiveMS/MStransitionsproteinssequences.Identifiedpeptideswerefilteredtoselectandpeptides,respectively.Subsequently,filterstoremoveonlyunitypicpeptidessatisfyingcurrentHPPGuidelinesv3.0standardRTcalibrationpeptidesandpeptideswith≥2missed(seeTable2),including≤1missedcleavage.Concurrently,ancleavageswereappliedforselectinguniquepeptidecandidates.insilicotrypticdigestionanalysiswasperformedforeachoftheDataProcessing36proteinswiththesamestringencyguidelinesappliedexceptazeromissedcleavagesrulewasimposed.ProteomecoverageSWATHextractionsbySkylineandPeakViewinitiallywentcorrespondingtotheseinsilicopeptidesforeachproteinwasthroughdatacleaningandfilteringbeforecomparisons.Allcalculatedtoderivethemaximumtheoreticalsequencedecoypeptides,RT-calibrationpeptidesandpeptideswithcoverageutilizingtrypticpeptides.morethantwomis-cleavageswereremoved.AllmodifiedpeptideswereremovedexceptcarbamidomethylatedcystineAsexpected,thenumberofidentifiedpeptidesobservedandandoxidizedmethioninecontainingpeptides.ThedataproteincoverageoftherecombinantproteinsbothdecreasedsubsequentlywasfurtherfilteredusingFDRcriteria.TwowhenHPPhigh-stringencyproteininferencemetricsweredifferentFDRcriteriawereusedfortheSWATHdataapplied,primarilyduetotheremovalofnested,non-unitypicextractedusingthetwodifferent(rPSLandplasma)libraries.and/or>1missedcleavagepeptides(Table2).WhenFortheSWATHdataextractedusingthemergedspectralcomparedtoinsilicodata,someproteins(i.e.,COQ,library,thedefaultPeakViewFDRcriterionwasused,i.e.,MMP2/3,KLK3,ITGB6,andS100A9)hadahighnumberpeptideswithatleastonesamplesatisfyingFDR<0.01wereofpeptideidentificationsaswellashighcoverage(seeTable2;kept.18,24FortheSWATHdataextractedusingtherPSL,acoverageobserved/expected).ThisindicatesthatmostmorestringentFDRcriterion(≥3replicateswithinagroupunitypicpeptidessatisfyinghigh-stringencyHPPMSGuide-havingFDR<0.01)wasappliedduetothepotentialforlesslinesv3.0requirementscouldbeobservedfromtherPSL.effectiveFDRcalculationresultingfromtheuseofasmallForsomeproteins,ahighernumberofpeptidesandspectrallibrary.coveragewereobservedexperimentallyatHPPstringencycomparedtoinsilicoexpectations.Thisislikelybecauseofthe■zeromissedcleavedruleappliedforallinsilicoanalysis,RESULTSwhereas≤1missedcleavagewasappliedforexperimentallyConstructionofaCancer-AssociatedRecombinantProteinobservedpeptides(i.e.,theDDAgeneratedrPSL).SpectralLibrary(rPSL)Furthermore,high-stringencyinsilicopredictionsuggestedDataprocessingandpeakfeatureextractionforidentificationthatonly2potentiallydetectablepeptidesmaybeavailableforofproteinsthroughSWATH-MSanalysisisdependentonthebothCXCL10(IP-10)andCXCL8(IL-8).Theplasma18,19qualityofpreviouslygeneratedDDAspectrallibraries.Theconcentrationsoftheseproteinsarelow(bothinpg/mLqualityandcoverageofspectrallibrarieshasbeenfoundtoberange)andneitherhavebeenpreviouslyidentifiedfromplasmadirectlyassociatedwiththeefficacyandscopeoffindingbyMS(Table1).OurinsilicoMSanalysisprovidesapossible91potentialcandidatesfromanySWATH-MSanalyses.explanationwhysomeplasmaproteinshavenotyetbeenInthisstudy,arPSLwasemployedtoassistinidentifyingidentifiedinhumanplasma.However,ournovelrPSLlowerabundancecancer-associatedplasmaproteinsbyapproachdetectspeptidesatthehighstringencylevel(3forSWATH-MS(Figure1).Intheinitialexperiment,ahigh-CXCL10and1forCXCL8)thathasallowedidentificationofqualityrPSLwithbroadcoverageforallofthe36full-lengththeseverylowabundanceplasmaproteinsfromnondepletedrecombinantproteins,eachofwhichhaspreviouslybeenCRCplasmasamples(seesectionbelow).reportedasaplasmacancerbiomarker,wasgenerated(TableTable2suggeststhatourrPSLapproachproducesamore1).TherPSLproteinswereselectedstrictlybasedonliteraturecomprehensivelibrarythatsignificantlyfacilitatesthechallengeexperimentalevidence(e.g.,MS,ELISA,WesternBlotting).ofdetectingpeptidesfromlowabundanceplasmaproteinsorAnalysisusingPeptideAtlasconfirmed9oftheseproteinsothercomplexbiospecimensbySWATH/DIA.(BTC,CXCL8,CXCL10,IL1B,IL6,ITGB6,TGFα,TNF,CRCPlasmaProteinBiomarkerIdentificationUsingtheTP53)hadnotpreviouslybeendetectedinhumanplasmabyrPSLSWATHApproachMS.Theplasmaconcentrationoftheseproteinshasbeenpreviouslyreportedtobe<10ng/mL(Table1),reflectingtheInordertoidentifyhowmanylowerabundancecancer-inherentchallengeoflowabundanceplasmaproteinassociatedplasmarPSLproteinswerepresentinCRCplasmas,identificationbyMSinthepresenceofHAPs.nondepletedplasmasfrompooledCRCpatients(n=5;seerPSLgenerationwasperformedusingDDA.Asexpected,allFigure1)wereexaminedbySWATH.Indetail,insteadof36recombinantproteinsweredetected,generallythroughausingSWATHinthetraditionalmanner(proteinquantifica-highnumberofpeptidesidentifiedathighconfidence(≥99%).tion),weusedittodetectpeptidesthatwouldordinarilynot2380https://doi.org/10.1021/acs.jproteome.0c00898J.ProteomeRes.2021,20,2374−2389

7JournalofProteomeResearchpubs.acs.org/jprArticleFigure2.AsuperimpositionofplasmaproteinconcentrationsofproteinsidentifiedfromtherecombinantproteinspectrallibrarycoupledwithSWATH-MSanalysis(rPSLSWATH)andtheplasmaproteinDDAspectrallibrary.TableS4containsalistofallidentifiedplasmaproteinsandpeptidesequencesusingtheDDAspectrallibrary.becapturedinstandardplasmaDDAexperiments.Thisnovelhypothesisthatlowabundancecancer-associatedplasmaapproachallowedlowabundanceproteinstobedetectedinaproteinspresentinthepg/mLrange(CEACAM5,CXCL8,singleexperiment,importantlywithoutdepletionorextensiveCXCL10,PDGFB,PTEN,TGFA,TP53)canbedetectedpeptidefractionation.Initially,aSWATH-MSdatasetwasusingnondepletedpatientplasmasamples.generatedfromeachCRCplasmasampleundertakenthroughTodemonstratetheabilityofrPSLSWATHtodetectlow5technicalreplicates.SWATH-MSdatasetswereanalyzedabundancecancer-associatedproteinsfromnondepletedCRCwithtwoindependentDIAanalysissoftwaretools,namely,plasma,wecomparedproteinidentificationsfromtherPSLPeakView(PV)andSkyline(SL).SWATHtoourroutineplasmaproteinidentificationmethodOfthe36rPSLbiomarkers,wereliablyidentified32proteins(i.e.,DDAshotgunproteomicsonHAP-depletedorpeptideusingPVand23proteinsusingSLinCRCplasmas.Inmostcases,higherpeptidecountswereobservedfromPVcomparedfractionatedplasma,referredtoas“plasmaproteinDDAtoSL(TableS3).AlthoughproteinsidentifiedfromPVspectrallibrary”,seeFigure1).coveredallproteinsfromSL,wenotedthathalfoftheSLTomaximizeplasmaproteinidentificationsfromDDAidentifiedpeptideswerenotcommontoPV.Thiswasexpectedshotgunruns,wecombinedhealthy/CRCplasmasamples(n=aseachsoftwarepackagereliesondifferentalgorithmsfor100)toensureproteinspresentinbothhealthyanddiseasedetectionandquantificationofuniquepeptidesandproteins.conditionswerepresent.Wesubsequentlyremovedthetop1492Similardiscordanceshavebeenobservedinapreviousstudy.HAPsusingAgilent’sMARS-14system.FollowingtrypticThepurposeofusingtwodifferentsoftwarepackageswasdigestionofMARS-14-depletedplasmas,reversed-phasedprimarilytotestwhethertherPSLSWATHapproachallowed7hydrophobicinteractionhighpHfractionationwasemployeddetectionoflow-abundancecancer-associatedproteinsthroughtoseparatepeptidesinto20fractions.ForproteineitherofthePVandSLplatforms.Ourstudyhasnotfocusedidentification,weusedanidenticalMSinstrumentandsettingsonacomprehensivecomparisonofthePVandSLsoftware92asfortherPSLSWATH.Wewereabletoidentifyatotaloftools,andreadersshouldrefertoapreviousarticleforsuchacomprehensivemulticenterbenchmarkingstudyinvolving742plasmaproteinsfrompooledhealthyorCRCplasmaslabel-freeproteomequantification.Tovisualizethedetectable(Figure2andTableS4forafulllistofidentifiedproteinsand7thresholdoflowabundanceproteinsusingtherPSLSWATHpeptidesequences).Comparedtoourpreviousstudy,weapproach,wesuperimposedthe32proteinsidentifiedontoaidentifiedanadditional229plasmaproteinsusingthemorehumanplasmaproteinconcentrationcurve(Figure2).ThisadvancedSCIEXTripleTOF6600MSinstrument(previouslyconcentrationcurvehasbeencreatedin-houseusingreported513proteinsidentifiedfromidenticalsamplesusingaSciexplasmaconcentrationsobtainedfromthePlasmaProteomeTripleTOF5600).786Database,PeptideAtlasandcomprehensiveliteratureOfthe32cancer-associatedplasmaproteinsidentifiedusingsearches.rPSLSWATH,12proteinscouldalsobeidentifiedinthisOneinterestingobservationfromthisdataisthatrPSLDDAplasmalibrary(Figure2).Interestingly,thereportedSWATHallowsidentificationofcandidatebiomarkersthatareconcentrationsofthese12proteinsweregenerallyhigherwidelyspreadacrosstheplasmaproteinconcentrationrangeabundanceproteins(Figure2,bluedotsonplasmaprotein(i.e.,highthroughtolowabundanceproteins,Figure2).Mostconcentrationcurve).Theremaining20proteinscouldimportantly,rPSLSWATHalloweddetectionofplasmaproteinsBTC,CXCL8,CXCL10,IL1B,IL6,ITGB6,TGFα,exclusivelyonlybeidentifiedbyournovelrPSLSWATHTNF,andTP53that(tothebestofourknowledge)havenotapproach,andtheseweregenerallylowerabundanceproteinsbeenpreviouslydetectedinhumanplasmausinganyMS(<10ng/mL;withexceptionofMMP3andKLK3;Figure2,technology.Furthermore,thedatastronglysupportthereddots).2381https://doi.org/10.1021/acs.jproteome.0c00898J.ProteomeRes.2021,20,2374−2389

8JournalofProteomeResearchpubs.acs.org/jprArticleFigure3.AcomparisonofidentifiedplasmaproteinsandpeptidesbetweenrecombinantproteinspectrallibrarycoupledwithSWATH-MSanalysis(rPSLSWATH)andSWATH-MSanalysisonthemergedlibrary.(a)NumberofdetectedpeptidesfromrPSLSWATHandSWATH-MSonmergedlibrarydemonstratingasimilartrendbutshowing3additionalproteinsidentifiedinthemergedapproach.(b)VenndiagramscomparingthenumberofcommonandunsharedidentifiedproteinsandpeptidesbetweenrPSLSWATHandSWATH-MSonthemergedlibrary.AlistofidentifiedpeptidesfromrPSLSWATHandmergedlibrarySWATHispresentedinTableS6.CRCPlasmaProteinBiomarkerIdentificationUsingawereidentifiedfor3proteins(BTC,CXCL8,TIMP2),andforMergedrPSLandHumanPlasmaProteinDDASpectraloneprotein(PTEN)ahighernumberofpeptides(4peptides)LibrarywereidentifiedfromrPSLSWATHcomparedtothemergedAlthoughSWATHanalysisisoneofthemostadvancedMSlibrarySWATH(3peptides)(Figure3a).Whencomparingtechnologies,thereremainsomeconcernsregardingincon-thetotalnumberofidentifiedpeptides,47peptidesweresistencyofdataanalysesfromasinglelibrarycomparedtocommonacrossbothmethods,and87peptideswereuniqueto93mergedlibraries.InevitableissuesaroundFDRcorrectionrPSL(Figure3b).AlistofidentifiedpeptidesfromrPSLarisewhenmergingdifferentsizedDDAlibrariesforSWATHSWATHandmergedlibrarySWATHispresentedinTableS6.analysis.TomitigateFDRconcernsresultingfromuseofaAlthoughtheoveralltrendofnumbersofpeptidesidentifiedsmallsinglelibrary(likerPSL),wemergedour36biomarkerwasrelativelyconsistentacrossbothapproaches,wewereableproteinrPSLlibrarywithastandardundepletedplasmaproteintocrediblyidentify3additionalproteins(ADAMDEC1,DDAlibrary(containinghigh-stringencydatafor742humanCST3,andPLAU)usingthemergedlibrary.Thisincreasesplasmaproteins).SWATHanalysiswasthenperformedwithidenticalexperimentalsettingsasusedabove(Figure1).ThethecoverageusingtherPSLfrom32(rPSL)to35(rPSL+mainreasonforusingamergedlibrarywastodetermineifdepletedplasmaDDAlibrary)outoftheoriginal36SWATHcontinuedtoidentifycandidatelowabundancerecombinantproteinsemployedinthisstudy.Interestingly,biomarkerproteinsinthepresenceofamuchmorecomplextheidentifiedpeptidesfromtheextrathreeproteinsoriginatedplasmalibrarybackground.fromtherPSLpartofthemergedlibrary,notthedepletedWecomparedlistsofidentifiedproteinsandpeptides(i.e.,plasmaDDAspectrallibrary(TableS7).Weassumethisisquantifiableproteinsandpeptides)derivedfromrPSLlikelyduetocomputationalissuesaroundFDR94andthisSWATHandSWATHMSanalysisusingthemergedlibrary.possibilityisdiscussedfurtherbelow.RTalignmentsbetweenlibraries,aswellastheCRCplasmaToincreasepeptideidentificationconfidence,wefinallySWATHMSdataset,werepeggedagainstpeptidesderivedappliedhigh-stringencyproteininferencecriteria(HPPfromtheabundantandubiquitousproteinsvitronectinand30Guidelinesv3.0)toalldetectedpeptidesusingthemergedalbumin.SWATHMSonthemergedlibraryidentifiedatotalrPSL+depletedplasmaDDAlibrary(seeFigure1fordetails).of519proteinssupportedby3187quantifiablepeptidesNotsurprisingly,thenumberofidentifiedpeptidessignificantly(Figure3,TableS5).Allthepreviouslyobservable32proteinsbiomarkersidentifiedusingtherPSLSWATHmethodcouldreducedforeachproteinafterhigh-stringencyfiltering(TablealsobeidentifiedusingamergedrPSL+depletedplasmaDDA3).Interestingly,CXCL8andTIMP2weredisqualifiedastheylibrary.Inmanycases,agreaternumberofpeptideswereonlyhadonepeptideidentified.TableS8containsdataontheidentifiedusingthemergedlibrarymethodcomparedwiththeproteinsandpeptidesequencesbeforeandafterapplyinghighrPSLSWATHsolely(Figure3a).Similarnumbersofpeptidesstringencyproteinidentificationcriteria.2382https://doi.org/10.1021/acs.jproteome.0c00898J.ProteomeRes.2021,20,2374−2389

9JournalofProteomeResearchpubs.acs.org/jprArticleTable3.DetectedPeptidesandProteinsfromNondepletedthisstudy,weaimedtoidentifyverylowabundancecancer-CRCPlasmabySWATH-MSAnalysisUsingtheMergedassociatedproteins(seeTable1fordetails)fromnondepletedaLibrary(rPSL+PlasmaProteinSpectralLibrary)CRCplasma,includingseveralnotdetectablebycurrentMSmethodologies,usingarPSLSWATHapproach.peptides#(HPPpeptides#(HPPbbAlthoughSWATH-MShasbeenavailableforproteingenenamesGuidelinesv3.0)genenamesGuidelinesv3.0)analysisandquantificationforadecade,thefundamentalITGAV22IL1B3hurdleofextractingconsistent,usefulandreliabledatafromPLG13IL63DIArunsremains.ItshouldbenotedthatherewehaveMMP911MIA3initiallydeliberatelyfocusedonproteinidentificationratherMMP210PDGFB3thanproteinquantification.WeappliedthesimplepremisethatMMP39TNFRSF1A3ifplasmaisarepositoryofmoleculesreflectingthebiologicalITGB19CST3297andphysiologicalstatusofthehumanbody,thenDIAMSTIMP17CXCL10298(IP10)(hereSWATH)shouldbeabletodetectallspectraincludingPFN17CXCL12(SDF-2lowabundanceproteinbiomarkers.Althoughtherearea1α)numberofwaystoinvestigatethis,includingmultiplelibraryCPQ7EGF2freeapproaches,99insilicospectrallibraries,100labeling,101CEACAM56BTC2spiking,102orsyntheticpeptideapproaches,103wechosetouseITGB66PTEN2alibraryapproachusingarecombinantproteinlibraryof36TP536S100A82low-mediumabundancesinceitwashopedthatamoreC1QC5S100A92comprehensivetrypticpeptiderepresentationforeachproteinEGFR5TGFA2couldbeachieved.PLAU5TNF2UsingarPSLpriortoSWATHonpatientsamples,wecADAMDEC14CXCL8(IL8)1achievedanaverageof70%coverageforeachproteinusingcKLK34TIMP21DDAanalysis,alevelofcoveragethatwouldnotbefeasible(atPLAUR4areasonablecost)withasyntheticpeptidelibraryorusingaThetableonlycontainsunitypicpeptidesthatsatisfiedthehigh-DDAmethodsonsimilarbiologicalplasmasamples.Thisstringencyproteininference/identificationcriteria,HPPGuidelinemakestherecombinantproteinDDAlibraryapproachmorebv3.0.SeeTableS8formoredetails.Non-nestedunitypicpeptidescomprehensiveforidentifyinglowabundanceproteinsinawith≥9aminoacids,≤1missedcleavage,≥2peptidesperprotein.SWATHexperiment.Additionally,routine,establishedandcProteinsthatwerenotqualifiedwithhighstringencyprotein17,104acceptedpipelinesfordataanalysiscouldbeappliedidentificationcriteria.leadingtomorereliableresults.InanySWATH-MSexperi-ment,themorecomprehensivethelibrary(resolution,peptide■DISCUSSIONnumber,proteincoverage)themoreaccuratewillbetheIdentificationofearlystage,lowabundancehumanplasmaidentifications.Toachieveacomprehensivelibrary,weranthecancerbiomarkersisanobjectiveofmanybiomarkerstudies.recombinantproteinsindiscretegroupsbasedonmolecularRecently,advancedtechnologieslikeliquidbiopsies(i.e.,weightgroupings(Figure1)toallowsuitablecoveragewithoutdetectingctDNA)95andSWATH/DIA-MS(i.e.,identifyingpeptidesfromsmallerproteinsoverwhelmingthosefromlargerandquantitatingcancer-associatedbiomarkerproteins)7areproteins.Thisallowedtheidentificationofahighnumberofmakingsuchanobjectivepotentiallyachievable.ThesepeptidesforeachproteinintherPSL,illustratinganaccurateadvancedtechnologiesfocusonadeeperunderstandingofandcomprehensiveDDAlibrary.plasmaforthesensitive,specificandaccuratemeasurementofThirty-twocancer-associatedplasmaproteinsweredetectedearlystagecancerbiomarkers.usingrPSLSWATH(from36),ofwhich20wereexclusivelyHowever,historicallymostcancer-associatedproteinbio-detectedwhencomparedtoaDDAplasmaproteinspectralmarkershavebeenreportedinpatientplasmaatparticularlylibrary(Figure2).Importantly,thereportedplasmaconcen-lowabundance.Indeed,thismaybemoreproblematicforearlytrationoftheseproteinswasbelow10ng/mL,and7proteinsstagecancerdetectionwhentumorsizeissmall,whilethewereactuallyinthepg/mLrange.Furthermore,33proteinsphysiologicalandimmuneresponsetothecancerisminimalwerereliablyidentifiedfromamergedlibrary(rPSL+MARS-andcancer-associatedbiomarkers(shedorleakedproteins)14depletedplasmaproteinDDAspectrallibrary)afterthewillalsobeoflowabundance.Inthecaseofprotein/peptideapplicationofhighstringencyproteinidentificationcriteria,30,31identification,thischallengeisexacerbatedbythehighHPPGuidelinev3.0(Table3).Theabilitytoreliablydynamicconcentrationrangeofproteinsfoundinplasma,detectlowerabundancedisease-relatedplasmaproteinshasmakingidentificationoflowabundanceproteinsbiomarkersapreviouslyonlybeenachievedwithmultidimensionalfractio-dauntingtask.Moststudiesattemptingtouncovercancer-nation,selectivemonitoring(orenrichment)oraffinity-basedassociatedplasmabiomarkersuseaseriesofdepletion,approaches.multidimensionalfractionationorsomeotherformofTherPSLSWATHallowedtheidentificationofwell-7,8,10,36enrichment.AlthoughMS-basedtechnologieshavedocumentedandclinicallysignificant(supportedbythebeensuggestedtobemorespecificandaccuratethanliterature)plasmaproteins.Numerousstudieshaveproposed3839antibody-basedtechniques,andareamenabletomultiplexCEAasalate-stageCRCbiomarker,aswellasforlungand40analysis,theyarenotyetcompatiblewithhigh-throughputgastriccancers.PlasmaIL6,CXCL8,andILB1havebeenmethodologiesandoftenfailtoidentifyverylowabundancealsoproposedasdiagnosticmarkersinCRC,brain,breast,oral48−50,58−60plasmaproteins.Conversely,antibody-basedtechnologiesandprostatecancers.Reportedplasmaconcen-96sufferfrombatchvariationissues,nonspecificbinding,astrationsofILB1andIL6areboth∼1ng/mL,andforCXCL8wellascostandreliabilityofdevelopingmultiplexassays.Inare∼14pg/mL.Ourstudyis,webelieve,thefirsttoshow2383https://doi.org/10.1021/acs.jproteome.0c00898J.ProteomeRes.2021,20,2374−2389

10JournalofProteomeResearchpubs.acs.org/jprArticledetectionofthesecytokinesinhumandiseaseplasmasusingsuitablemethodastherPSLlibraryneedsonlytobedeveloped25MS.Furthermore,rPSLSWATHwasabletoidentifyCRConce,andunlikeapredictedlibrary(asdonebyPROSIT),biomarkersITGB6andPLAURimplicatedinourowntheselibrarieshaverealempiricalevidence.Hence,rPSLcan7,26−28,105previousstudies.Thereportedplasmaconcentrationsbetailoredtothespecificproteindetectionpanel,andonceofbothITGB6andPLAURare∼2ng/mLandagainthisbuiltcanbepermanentlyused.Thus,panelsofhundreds,orstudyis,toourknowledge,thefirsttodemonstrateplasmaeventhousands,ofproteinsandresultanttrypticpeptidescanITGB6detectionusingMS.PLAURexpressionintumorbedetectedsimultaneouslyinahigh-throughputmanner.tissuesandplasmahasbeenrecognizedasapotentialAsemphasizedinthemethods,thisexperimentfocused106biomarkerformanycancertypes,includingCRC.Notably,solelyonidentification,usingclinicalsamples,withanPLAURmeasurementontumorcellsurfacesanddetectionofemphasisonusingmergedmultipleSWATHlibrariesforsoluble-uPAR(suPAR)(cleaveduPARisoformsreleasedfromconfirmation.AdrawbackofsuchanapproachisthatthecellsurfacecontainingdomainsD1,D2+D3,orquantitativedatamaynotbecompletelyreliableasdifferences18,24D1+D2+D3)inplasmahavebeenrecognizedasprognosticinanalytesandidentificationswillinevitablybepresent.73,104WehaveextensivelyinvestigatedtheeffectofusingdifferentindicatorsofCRCsurvival.However,suchstudiesused18,24antibody-basedtechnologieswhichcanhaveissueswithbothlibrariesasinotherworksthathavenoteddifferencesin96analytedetectionandquantitation.Tojustifytheuseofthenonspecificbindingandbatchvariation.Wecontendthatdevelopinghigh-throughputtargetedMSprognostictoolsrPSLSWATHapproachforquantitationandsubsequentemployinganovelrPSLapproachwillbeofsignificantbenefitvalidation,anumberofadditionalexperimentswouldbeacrossmanyclinicalsettings.Collectively,identificationandrequiredincludingtheuseofspikedpeptides(labeledandquantificationoflowabundancecancer-associatedproteinsnativeunlabeledforms)atknownquantitiesincludedwithfromnondepletedornonfractionatedplasmaallowsamorenondepletedplasma,alargerstagedcancerpatientsample102cohort(toallowaccuratestatisticalconfidence),comprehen-seamlesstransitiontopotentialclinicalapplications,eliminatestheriskofdepletionofunintendednontargetedsiveinformaticsandstatisticsespeciallyaroundFDR(whichis107heretounappreciatedonsuchanapproach).proteinsandservestodemonstratetheefficiencyofSWATH-MS.Inconclusion,thisstudyusedanrPSLSWATHapproachtoAlthoughournovelrPSLSWATHapproachhassubstantialidentifylowerabundancecancer-associatedproteinsfrombenefitsforproteomicsapplications,thereareissueswhichnondepletedCRCplasmas.WewereabletodemonstratethemustbeconsideredaroundFDRcorrectionswhenmergingpremisethatthisnovelapproachcanprobedeeperintothedifferentlysizedDDAlibraries.Inthisstudy,weaddressedtheplasmaproteomecomparedtostandardDDAshotgunFDRconcernsduetotheuseofasmalllibrarybymergingtwoproteomicsorDIA/SWATHwhereDDAlibrariesarelibraries(rPSLandplasmaproteinDDAlibrary).SWATHgeneratedfrombiologicalsamples.UsingrPSLSWATH,weanalysisusingthismergedlibraryresultedinthreeadditionalwereabletosee8additionalproteinsthathaveneverplasmaproteinidentifications(ADAMDEC1,CST3,andpreviouslybeenobservedbyMSathigh-stringencyproteinPLAU).However,detectedquantifiablepeptidesfortheseinference.TheimplicationofthisstudyisthatMStechnologiesthreeproteinswerederivedfromtherPSLpartofthemerge,canreliablyachievepicogramdetectiononnondepletednotfromtheMARS-14depletedplasmaproteinDDAlibrary.plasma(currentlythoughttobeobtainableonlyusingmoreWerecognizethattherPSLisarelativelysmalllibrarysensitiveantibody-basedaffinitymethods).Wecontendthatconstructedusingonly36recombinantproteinsand1435rPSLSWATHapproachescanallowaccurate,reliable,andpeptideidentifications.SWATHanalysisat1%FDRprovidedreadilyadaptableclinicalmeasurementofmultiplelow32proteinidentificationssupportedby134(or9.3%)abundanceplasmabiomarkers(panels)simultaneouslyinaquantifiablepeptidesfromnondepletedCRCplasma.Insingleworkflow.contrast,themergedlibrarycontained762proteinsand33295peptides,andSWATHanalysis(1%FDR)resultedin■ASSOCIATEDCONTENT519proteinidentificationswith3187(or9.5%)quantifiable*sıSupportingInformationpeptides.AlthoughtheratioofquantifiablepeptidesbetweenTheSupportingInformationisavailablefreeofchargeattherPSL(smaller)andmergedlibrary(larger)wassimilar,thehttps://pubs.acs.org/doi/10.1021/acs.jproteome.0c00898.actualpeptidecontentwasdifferentduetotheapplicationofdifferentFDRrequirementsforthedifferentlibraries,aswellasSITableofContents(PDF)thedifferentlibrarysize.Weassumedthat,inrPSL,theTableS1:Skylinepeptideandtransitionsettings(PDF)peptides(forADAMDEC1,CST3,andPLAU)mayhavebeenTableS2:Identifiedproteins/peptidesfromDDAeliminatedasfalsepositives(i.e.,notpassedatthe1%FDRcutrecombinantproteinspectrallibrary(rPSL)(XLSX)offforatleastthreereplicates).However,whenthelibrariesTableS3:Identifiedproteins/peptidesfromrPSLweremerged,thenumberoffalsepositivepeptidesfromtheSWATHanalysisusingPeakViewandSkyline(XLSX)largerlibrarymayhavebeenoverwhelmedsuchthateliminatedTableS4:Identifiedproteins/peptidesfromhumanpeptidesinrPSLmayhavebeenacceptedastruepositivesinplasmaDDAlibrary(XLSX)thelargermergedlibrary.TableS5:Identifiedproteins/peptidesfrommergedDDAexperimentsoftensufferfromstochasticselectionof20librarySWATHanalysis(XLSX)precursorionsforMS/MSfragmentation,andthisespeciallyappliestoclinicalplasmastudiesduetothecomplexityoftheTableS6:AlistofidentifiedpeptidesfromrPSLsamplesused.SWATH/DIAstudiesinvolvingDDAlibrarySWATHandmergedlibrarySWATH(XLSX)buildingalsosuffersimilarstochasticselectionissues.IntheTableS7:Peptides(forADAMDEC1,CST3,andcaseofstudieswhereaspecificpanelofproteinsneedstobePLAU)identifiedfromrPSL,humanplasmaDDAmonitoredroutinelyinamultiplexedmanner,rPSLmaybealibrary,andmergedlibrarySWATHanalysis(XLSX)2384https://doi.org/10.1021/acs.jproteome.0c00898J.ProteomeRes.2021,20,2374−2389

11JournalofProteomeResearchpubs.acs.org/jprArticleTableS8:IdentifiedproteinsandpeptidesfrommergedKSK,AM,SA,JXW,DP,HRC,GJG,andECNpreparedandlibrarySWATH,beforeandafterapplyinghighrevisedfiguresandtables.Allauthorscontributedtowriting,stringencyproteinidentificationcriteria(XLSX)review,andrevisionofeachmanuscriptversion.■NotesAUTHORINFORMATIONTheauthorsdeclarenocompetingfinancialinterest.CorrespondingAuthorsMassspectrometrydataisavailablethroughtheProteomeX-SeongBeomAhn−DepartmentofBiomedicalSciences,changeconsortiumviathePRIDEpartnerrepositorywiththeFacultyofMedicineandHealthSciences,MacquariedatasetidentifierPXD022361.University,MacquariePark,NSW2109,Australia;orcid.org/0000-0001-5907-3544;Phone:+6129850■ACKNOWLEDGMENTS2717;Email:charlie.ahn@mq.edu.auMarkS.Baker−DepartmentofBiomedicalSciences,FacultyTheauthorsacknowledgeandthanktheVictorianCancerofMedicineandHealthSciences,MacquarieUniversity,BiobankforprovidingCRCpatientEDTA-plasmasamples.MacquariePark,NSW2109,Australia;orcid.org/0000-ThisstudywassupportedbytheCancerInstituteNSWECR0001-5858-4035;Phone:+61298508211;fellowship15/ECF/1-38(SBA),CancerCouncilNSWRG19-Email:mark.baker@mq.edu.au04(MSB,SBA,ECN),NHMRCprojectgrant1010303(MSB,ECN),“FightontheBeaches”(MSB,SBA,ECN,SA),SydneyAuthorsVitalCINSWTranslationalCancerResearchCentregrantKarthikS.Kamath−AustralianProteomeAnalysisFacility(MSB,SBA,SA),andiMQRESfundingfromMacquarie(APAF),DepartmentofMolecularSciences,FacultyofUniversity(SA).ScienceandEngineering,MacquarieUniversity,MacquariePark,NSW2109,Australia■REFERENCESAbidaliMohamedali−DepartmentofMolecularSciences,(1)Dakubo,G.D.AdvancedTechnologiesforBodyFluidFacultyofScienceandEngineering,MacquarieUniversity,BiomarkerAnalyses.InCancerBiomarkersinBodyFluids;Springer,MacquariePark,NSW2109,Australia2016;pp55−74.ZainabNoor−ProCan,Children’sMedicalResearchInstitute,(2)Ridker,P.M.;Rifai,N.;Stampfer,M.J.;Hennekens,C.H.TheUniversityofSydney,Westmead,Newtown,NSW2042,Plasmaconcentrationofinterleukin-6andtheriskoffutureAustraliamyocardialinfarctionamongapparentlyhealthymen.CirculationJemmaX.Wu−AustralianProteomeAnalysisFacility2000,101(15),1767−72.(APAF),DepartmentofMolecularSciences,Facultyof(3)Geyer,P.E.;Holdt,L.M.;Teupser,D.;Mann,M.RevisitingScienceandEngineering,MacquarieUniversity,Macquariebiomarkerdiscoverybyplasmaproteomics.Mol.Syst.Biol.2017,13(9),942.Park,NSW2109,Australia;orcid.org/0000-0001-8578-(4)Nice,E.C.Theseparationsciences,thefrontendtoproteomics:8455Anhistoricalperspective.BiomedChromatogr.2021,35(1),e4995.DanaPascovici−AustralianProteomeAnalysisFacility(5)Chantaraamporn,J.;Champattanachai,V.;Khongmanee,A.;(APAF),DepartmentofMolecularSciences,FacultyofVerathamjamras,C.;Prasongsook,N.;Mingkwan,K.;Luevisadpibul,ScienceandEngineering,MacquarieUniversity,MacquarieV.;Chutipongtanate,S.;Svasti,J.GlycoproteomicAnalysisRevealsPark,NSW2109,Australia;orcid.org/0000-0002-3266-AberrantExpressionofComplementC9andFibronectininthe4851PlasmaofPatientswithColorectalCancer.Proteomes2020,8(3),26.SubashAdhikari−DepartmentofBiomedicalSciences,(6)Clark,D.J.;Dhanasekaran,S.M.;Petralia,F.;Pan,J.;Song,X.;FacultyofMedicineandHealthSciences,MacquarieHu,Y.;daVeigaLeprevost,F.;Reva,B.;Lih,T.M.;Chang,H.Y.;University,MacquariePark,NSW2109,Australia;Ma,W.;Huang,C.;Ricketts,C.J.;Chen,L.;Krek,A.;Li,Y.;Rykunov,D.;Li,Q.K.;Chen,L.S.;Ozbek,U.;Vasaikar,S.;Wu,Y.;orcid.org/0000-0001-5945-7804Yoo,S.;Chowdhury,S.;Wyczalkowski,M.A.;Ji,J.;Schnaubelt,M.;HarishR.Cheruku−DepartmentofBiomedicalSciences,Kong,A.;Sethuraman,S.;Avtonomov,D.M.;Ao,M.;Colaprico,A.;FacultyofMedicineandHealthSciences,MacquarieCao,S.;Cho,K.C.;Kalayci,S.;Ma,S.;Liu,W.;Ruggles,K.;University,MacquariePark,NSW2109,AustraliaCalinawan,A.;Gümü,Z.H.;Geiszler,D.;Kawaler,E.;Teo,G.C.;GillesJ.Guillemin−DepartmentofBiomedicalSciences,Wen,B.;Zhang,Y.;Keegan,S.;Li,K.;Chen,F.;Edwards,N.;FacultyofMedicineandHealthSciences,MacquariePierorazio,P.M.;Chen,X.S.;Pavlovich,C.P.;Hakimi,A.A.;University,MacquariePark,NSW2109,AustraliaBrominski,G.;Hsieh,J.J.;Antczak,A.;Omelchenko,T.;Lubinski,J.;MatthewJ.McKay−AustralianProteomeAnalysisFacilityWiznerowicz,M.;Linehan,W.M.;Kinsinger,C.R.;Thiagarajan,M.;(APAF),DepartmentofMolecularSciences,FacultyofBoja,E.S.;Mesri,M.;Hiltke,T.;Robles,A.I.;Rodriguez,H.;Qian,ScienceandEngineering,MacquarieUniversity,MacquarieJ.;Fenyö,D.;Zhang,B.;Ding,L.;Schadt,E.;Chinnaiyan,A.M.;Park,NSW2109,AustraliaZhang,Z.;Omenn,G.S.;Cieslik,M.;Chan,D.W.;Nesvizhskii,A.I.;Wang,P.;Zhang,H.;etal.IntegratedProteogenomicCharacter-EdouardC.Nice−DepartmentofBiochemistryandizationofClearCellRenalCellCarcinoma.Cell2019,179(4),964−MolecularBiology,FacultyofMedicine,NursingandHealth983.Sciences,MonashUniversity,Clayton,VIC3800,Australia(7)Ahn,S.B.;Sharma,S.;Mohamedali,A.;Mahboob,S.;Redmond,Completecontactinformationisavailableat:W.J.;Pascovici,D.;Wu,J.X.;Zaw,T.;Adhikari,S.;Vaibhav,V.;https://pubs.acs.org/10.1021/acs.jproteome.0c00898Nice,E.C.;Baker,M.S.Potentialearlyclinicalstagecolorectalcancerdiagnosisusingaproteomicsbloodtestpanel.Clin.Proteomics2019,16,34.AuthorContributions(8)Yadav,A.K.;Bhardwaj,G.;Basak,T.;Kumar,D.;Ahmad,S.;SBAandMSBdesignedallexperiments.SBA,KSK,AM,SA,Priyadarshini,R.;Singh,A.K.;Dash,D.;Sengupta,S.AsystematicandMJMperformedexperiments.SBA,KSK,AM,ZN,JXW,analysisofelutedfractionofplasmapostimmunoaffinitydepletion:andDPperformedMSdataandstatisticalanalyses.SBA,MSB,implicationsinbiomarkerdiscovery.PLoSOne2011,6(9),e24442.2385https://doi.org/10.1021/acs.jproteome.0c00898J.ProteomeRes.2021,20,2374−2389

12JournalofProteomeResearchpubs.acs.org/jprArticle(9)Nice,E.C.;Rothacker,J.;Weinstock,J.;Lim,L.;Catimel,B.UseSWATH-MassSpectrometrywithLargePeptideReferenceLibraries.ofmultidimensionalseparationprotocolsforthepurificationoftraceProteomics2017,17,1700174.componentsincomplexbiologicalsamplesforproteomicsanalysis.J.(25)Gessulat,S.;Schmidt,T.;Zolg,D.P.;Samaras,P.;Schnatbaum,ChromatogrA2007,1168(1−2),190−210discussion189.K.;Zerweck,J.;Knaute,T.;Rechenberger,J.;Delanghe,B.;Huhmer,(10)Seong,Y.;Yoo,Y.S.;Akter,H.;Kang,M.J.SamplepreparationA.;Reimer,U.;Ehrlich,H.C.;Aiche,S.;Kuster,B.;Wilhelm,M.fordetectionoflowabundanceproteinsinhumanplasmausingultra-Prosit:proteome-widepredictionofpeptidetandemmassspectrabyhighperformanceliquidchromatographycoupledwithhighlydeeplearning.Nat.Methods2019,16(6),509−518.accuratemassspectrometry.J.Chromatogr.B:Anal.Technol.Biomed.(26)Ahn,S.B.;Chan,C.;Dent,O.F.;Mohamedali,A.;Kwun,S.Y.;LifeSci.2017,1060,272−280.Clarke,C.;Fletcher,J.;Chapuis,P.H.;Nice,E.C.;Baker,M.S.(11)Ahn,S.-B.;Khan,A.Detectionandquantitationoftwenty-Epithelialandstromalcellurokinaseplasminogenactivatorreceptorsevencytokines,chemokinesandgrowthfactorspre-andpost-highexpressiondifferentiallycorrelateswithsurvivalinrectalcancerstagesabundanceproteindepletioninhumanplasma.EuPaOpenProteomicsBandCpatients.PLoSOne2015,10(2),e0117786.2014,3,78−84.(27)Ahn,S.B.;Mohamedali,A.;Chan,C.;Fletcher,J.;Kwun,S.Y.;(12)Lim,S.Y.;Lee,J.H.;Welsh,S.J.;Ahn,S.B.;Breen,E.;Khan,Clarke,C.;Dent,O.F.;Chapuis,P.H.;Nice,E.;Baker,M.S.A.;Carlino,M.S.;Menzies,A.M.;Kefford,R.F.;Scolyer,R.A.;Correlationsbetweenintegrinανβ6expressionandclinico-patho-Long,G.V.;Rizos,H.Evaluationoftwohigh-throughputproteomiclogicalfeaturesinstageBandstageCrectalcancer.PLoSOne2014,9technologiesforplasmabiomarkerdiscoveryinimmunotherapy-(5),e97248.treatedmelanomapatients.BiomarkRes.2017,5,32.(28)Ahn,S.B.;Mohamedali,A.;Pascovici,D.;Adhikari,S.;Sharma,(13)Tu,C.;Rudnick,P.A.;Martinez,M.Y.;Cheek,K.L.;Stein,S.S.;Nice,E.C.;Baker,M.S.ProteomicsRevealsCell-SurfaceE.;Slebos,R.J.;Liebler,D.C.DepletionofabundantplasmaproteinsUrokinasePlasminogenActivatorReceptorExpressionImpactsMostandlimitationsofplasmaproteomics.J.ProteomeRes.2010,9(10),HallmarksofCancer.Proteomics2019,19,1900026.4982−91.(29)Mahboob,S.;Ahn,S.B.;Cheruku,H.R.;Cantor,D.;Rennel,(14)Keshishian,H.;Burgess,M.W.;Gillette,M.A.;Mertins,P.;E.;Fredriksson,S.;Edfeldt,G.;Breen,E.J.;Khan,A.;Mohamedali,Clauser,K.R.;Mani,D.R.;Kuhn,E.W.;Farrell,L.A.;Gerszten,R.A.;Muktadir,M.G.;Ranganathan,S.;Tan,S.H.;Nice,E.;Baker,M.E.;Carr,S.A.Multiplexed,QuantitativeWorkflowforSensitiveS.AnovelmultiplexedimmunoassayidentifiesCEA,IL-8andBiomarkerDiscoveryinPlasmaYieldsNovelCandidatesforEarlyprolactinasprospectivemarkersforDukes’stagesA-DcolorectalMyocardialInjury.Mol.CellProteomics2015,14(9),2375−93.cancers.Clin.Proteomics2015,12(1),10.(15)Geyer,P.E.;Kulak,N.A.;Pichler,G.;Holdt,L.M.;Teupser,(30)Deutsch,E.W.;Lane,L.;Overall,C.M.;Bandeira,N.;Baker,D.;Mann,M.PlasmaProteomeProfilingtoAssessHumanHealthM.S.;Pineau,C.;Moritz,R.L.;Corrales,F.;Orchard,S.;VanEyk,J.andDisease.CellSyst2016,2(3),185−95.E.;Paik,Y.K.;Weintraub,S.T.;Vandenbrouck,Y.;Omenn,G.S.(16)Schwenk,J.M.;Omenn,G.S.;Sun,Z.;Campbell,D.S.;Baker,HumanProteomeProjectMassSpectrometryDataInterpretationM.S.;Overall,C.M.;Aebersold,R.;Moritz,R.L.;Deutsch,E.W.Guidelines3.0.J.ProteomeRes.2019,18(12),4108−4116.TheHumanPlasmaProteomeDraftof2017:BuildingontheHuman(31)Adhikari,S.;Nice,E.C.;Deutsch,E.W.;Lane,L.;Omenn,G.PlasmaPeptideAtlasfromMassSpectrometryandComplementaryS.;Pennington,S.R.;Paik,Y.K.;Overall,C.M.;Corrales,F.J.;Assays.J.ProteomeRes.2017,16(12),4299−4310.Cristea,I.M.;VanEyk,J.E.;Uhlén,M.;Lindskog,C.;Chan,D.W.;(17)Ludwig,C.;Gillet,L.;Rosenberger,G.;Amon,S.;Collins,B.Bairoch,A.;Waddington,J.C.;Justice,J.L.;LaBaer,J.;Rodriguez,H.;C.;Aebersold,R.Data-independentacquisition-basedSWATH-MSHe,F.;Kostrzewa,M.;Ping,P.;Gundry,R.L.;Stewart,P.;Srivastava,forquantitativeproteomics:atutorial.Mol.Syst.Biol.2018,14(8),S.;Srivastava,S.;Nogueira,F.C.S.;Domont,G.B.;Vandenbrouck,e8126.Y.;Lam,M.P.Y.;Wennersten,S.;Vizcaino,J.A.;Wilkins,M.;(18)Wu,J.X.;Song,X.;Pascovici,D.;Zaw,T.;Care,N.;Krisp,C.;Schwenk,J.M.;Lundberg,E.;Bandeira,N.;Marko-Varga,G.;Molloy,M.P.SWATHMassSpectrometryPerformanceUsingWeintraub,S.T.;Pineau,C.;Kusebauch,U.;Moritz,R.L.;Ahn,S.B.;ExtendedPeptideMS/MSAssayLibraries.Mol.CellProteomics2016,Palmblad,M.;Snyder,M.P.;Aebersold,R.;Baker,M.S.Ahigh-15(7),2501−14.stringencyblueprintofthehumanproteome.Nat.Commun.2020,11(19)Schubert,O.T.;Gillet,L.C.;Collins,B.C.;Navarro,P.;(1),5301.Rosenberger,G.;Wolski,W.E.;Lam,H.;Amodei,D.;Mallick,P.;(32)Liu,T.;Qian,W.J.;Gritsenko,M.A.;Xiao,W.;Moldawer,L.MacLean,B.;Aebersold,R.Buildinghigh-qualityassaylibrariesforL.;Kaushal,A.;Monroe,M.E.;Varnum,S.M.;Moore,R.J.;Purvine,targetedanalysisofSWATHMSdata.Nat.Protoc.2015,10(3),S.O.;Maier,R.V.;Davis,R.W.;Tompkins,R.G.;Camp,D.G.,2nd;426−41.(20)Gillet,L.C.;Navarro,P.;Tate,S.;Rost,H.;Selevsek,N.;Smith,R.D.;Inflammation;theHostResponsetoInjuryLargeScaleReiter,L.;Bonner,R.;Aebersold,R.TargeteddataextractionoftheCollaborativeResearch,P..HighdynamicrangecharacterizationofMS/MSspectrageneratedbydata-independentacquisition:anewthetraumapatientplasmaproteome.Mol.CellProteomics2006,5conceptforconsistentandaccurateproteomeanalysis.Mol.Cell(10),1899−1913.Proteomics2012,11(6),O111.016717.(33)Kume,H.;Muraoka,S.;Kuga,T.;Adachi,J.;Narumi,R.;(21)Noor,Z.;Ahn,S.B.;Baker,M.S.;Ranganathan,S.;Watanabe,S.;Kuwano,M.;Kodera,Y.;Matsushita,K.;Fukuoka,J.;Mohamedali,A.Massspectrometry-basedproteinidentificationinMasuda,T.;Ishihama,Y.;Matsubara,H.;Nomura,F.;Tomonaga,T.proteomics-areview.BriefingsBioinf.2020,DOI:10.1093/bib/Discoveryofcolorectalcancerbiomarkercandidatesbymembranebbz163.proteomicanalysisandsubsequentverificationusingselectedreaction(22)Zi,J.;Zhang,S.;Zhou,R.;Zhou,B.;Xu,S.;Hou,G.;Tan,F.;monitoring(SRM)andtissuemicroarray(TMA)analysis.Mol.CellWen,B.;Wang,Q.;Lin,L.;Liu,S.ExpansionoftheionlibraryforProteomics2014,13(6),1471−84.miningSWATH-MSdatathroughfractionationproteomics.Anal.(34)Jiang,W.;Huang,R.;Duan,C.;Fu,L.;Xi,Y.;Yang,Y.;Yang,Chem.2014,86(15),7242−6.W.M.;Yang,D.;Yang,D.H.;Huang,R.P.Identificationoffive(23)Rosenberger,G.;Koh,C.C.;Guo,T.;Rost,H.L.;Kouvonen,serumproteinmarkersfordetectionofovariancancerbyantibodyP.;Collins,B.C.;Heusel,M.;Liu,Y.;Caron,E.;Vichalkovski,A.;arrays.PLoSOne2013,8(10),e76795.Faini,M.;Schubert,O.T.;Faridi,P.;Ebhardt,H.A.;Matondo,M.;(35)Ueda,K.;Tatsuguchi,A.;Saichi,N.;Toyama,A.;Tamura,K.;Lam,H.;Bader,S.L.;Campbell,D.S.;Deutsch,E.W.;Moritz,R.L.;Furihata,M.;Takata,R.;Akamatsu,S.;Igarashi,M.;Nakayama,M.;Tate,S.;Aebersold,R.Arepositoryofassaystoquantify10,000Sato,T.A.;Ogawa,O.;Fujioka,T.;Shuin,T.;Nakamura,Y.;humanproteinsbySWATH-MS.Sci.Data2014,1,140031.Nakagawa,H.Plasmalow-molecular-weightproteomeprofiling(24)Wu,J.X.;Pascovici,D.;Ignjatovic,V.;Song,X.;Krisp,C.;identifiedneuropeptide-Yasaprostatecancerbiomarkerpolypeptide.Molloy,M.P.ImprovingProteinDetectionConfidenceUsingJ.ProteomeRes.2013,12(10),4497−506.2386https://doi.org/10.1021/acs.jproteome.0c00898J.ProteomeRes.2021,20,2374−2389

13JournalofProteomeResearchpubs.acs.org/jprArticle(36)Whiteaker,J.R.;Zhang,H.;Eng,J.K.;Fang,R.;Piening,B.D.;(49)Ilhan-Mutlu,A.;Wagner,L.;Widhalm,G.;Wohrer,A.;Bartsch,Feng,L.C.;Lorentzen,T.D.;Schoenherr,R.M.;Keane,J.F.;S.;Czech,T.;Heinzl,H.;Leutmezer,F.;Prayer,D.;Marosi,C.;Base,Holzman,T.;Fitzgibbon,M.;Lin,C.;Zhang,H.;Cooke,K.;Liu,T.;W.;Preusser,M.ExploratoryinvestigationofeightcirculatingplasmaCamp,D.G.,2nd;Anderson,L.;Watts,J.;Smith,R.D.;McIntosh,M.markersinbraintumorpatients.NeurosurgRev.2013,36(1),45−55W.;Paulovich,A.G.Head-to-headcomparisonofserumfractionationdiscussion55−56.techniques.J.ProteomeRes.2007,6(2),828−36.(50)Todorovic-Rakovic,N.;Milovanovic,J.Interleukin-8inbreast(37)Yan,B.;Chen,B.;Min,S.;Gao,Y.;Zhang,Y.;Xu,P.;Li,C.;cancerprogression.J.InterferonCytokineRes.2013,33(10),563−70.Chen,J.;Luo,G.;Liu,C.iTRAQ-basedComparativeSerum(51)Narita,D.;Seclaman,E.;Anghel,A.;Ilina,R.;Cireap,N.;ProteomicAnalysisofProstateCancerPatientswithorwithoutNegru,S.;Sirbu,I.O.;Ursoniu,S.;Marian,C.AlteredlevelsofplasmaBoneMetastasis.J.Cancer2019,10(18),4165−4177.chemokinesinbreastcancerandtheirassociationwithclinicaland(38)Christensen,I.J.;Brunner,N.;Dowell,B.;Davis,G.;Nielsen,pathologicalcharacteristics.Neoplasma2016,63(1),141−9.H.J.;Newstead,G.;King,D.PlasmaTIMP-1andCEAasMarkersfor(52)Guldbrandsen,A.;Vethe,H.;Farag,Y.;Oveland,E.;Garberg,DetectionofPrimaryColorectalCancer:AProspectiveValidationH.;Berle,M.;Myhr,K.M.;Opsahl,J.A.;Barsnes,H.;Berven,F.S.StudyIncludingSymptomaticandNon-symptomaticIndividuals.In-depthcharacterizationofthecerebrospinalfluid(CSF)proteomeAnticancerRes.2015,35(9),4935−4941.displayedthroughtheCSFproteomeresource(CSF-PR).Mol.Cell(39)Djureinovic,D.;Ponten,V.;Landelius,P.;AlSayegh,S.;Proteomics2014,13(11),3152−63.Kappert,K.;Kamali-Moghaddam,M.;Micke,P.;Stahle,E.Multiplex(53)Lavaee,F.;Zare,S.;Mojtahedi,Z.;Malekzadeh,M.;Khademi,plasmaproteinprofilingidentifiesnovelmarkerstodiscriminateB.;Ghaderi,A.SerumCXCL12,butnotCXCR4,IsAssociatedwithpatientswithadenocarcinomaofthelung.BMCCancer2019,19(1),HeadandNeckSquamousCellCarcinomas.AsianPac.J.CancerPrev.741.2018,19(4),901−904.(40)Shen,Q.;Polom,K.;Williams,C.;deOliveira,F.M.S.;(54)Lukaszewicz-Zajac,M.;Mroczko,B.;Kozlowski,M.;Guergova-Kuras,M.;Lisacek,F.;Karlsson,N.G.;Roviello,F.;Szmitkowski,M.TheSerumConcentrationsofChemokineKamali-Moghaddam,M.AtargetedproteomicsapproachrevealsaCXCL12andItsSpecificReceptorCXCR4inPatientswithserumproteinsignatureasdiagnosticbiomarkerforresectablegastricEsophagealCancer.Dis.Markers2016,2016,7963895.cancer.EBioMedicine2019,44,322−333.(55)Blanco-Prieto,S.;Vazquez-Iglesias,L.;Rodriguez-Girondo,M.;(41)Braga-Lagache,S.;Buchs,N.;Iacovache,M.I.;Zuber,B.;Barcia-Castro,L.;Fernandez-Villar,A.;Botana-Rial,M.I.;Rodriguez-Jackson,C.B.;Heller,M.RobustLabel-free,QuantitativeProfilingofBerrocal,F.J.;delaCadena,M.P.Serumcalprotectin,CD26andCirculatingPlasmaMicroparticle(MP)AssociatedProteins.Mol.CellEGFtoestablishapanelforthediagnosisoflungcancer.PLoSOneProteomics2016,15(12),3640−3652.2015,10(5),e0127318.(42)Geyer,P.E.;WewerAlbrechtsen,N.J.;Tyanova,S.;Grassl,N.;(56)Omenn,G.S.;States,D.J.;Adamski,M.;Blackwell,T.W.;Iepsen,E.W.;Lundgren,J.;Madsbad,S.;Holst,J.J.;Torekov,S.S.;Menon,R.;Hermjakob,H.;Apweiler,R.;Haab,B.B.;Simpson,R.J.;Mann,M.ProteomicsrevealstheeffectsofsustainedweightlossonEddes,J.S.;Kapp,E.A.;Moritz,R.L.;Chan,D.W.;Rai,A.J.;thehumanplasmaproteome.Mol.Syst.Biol.2016,12(12),901.Admon,A.;Aebersold,R.;Eng,J.;Hancock,W.S.;Hefta,S.A.;(43)Lee,J.H.;Cho,H.S.;Lee,J.J.;Jun,S.Y.;Ahn,J.H.;Min,J.S.;Meyer,H.;Paik,Y.K.;Yoo,J.S.;Ping,P.;Pounds,J.;Adkins,J.;Qian,Yoon,J.Y.;Choi,M.H.;Jeon,S.J.;Lim,J.H.;Jung,C.R.;Kim,D.X.;Wang,R.;Wasinger,V.;Wu,C.Y.;Zhao,X.;Zeng,R.;Archakov,S.;Kim,H.T.;Factor,V.M.;Lee,Y.H.;Thorgeirsson,S.S.;Kim,C.A.;Tsugita,A.;Beer,I.;Pandey,A.;Pisano,M.;Andrews,P.;H.;Kim,N.S.PlasmaglutamatecarboxypeptidaseisanegativeTammen,H.;Speicher,D.W.;Hanash,S.M.OverviewoftheHUPOregulatorinlivercancermetastasis.Oncotarget2016,7(48),79774−PlasmaProteomeProject:resultsfromthepilotphasewith3579786.collaboratinglaboratoriesandmultipleanalyticalgroups,generatinga(44)Chen,R.;Mias,G.I.;Li-Pook-Than,J.;Jiang,L.;Lam,H.Y.;coredatasetof3020proteinsandapublicly-availabledatabase.Chen,R.;Miriami,E.;Karczewski,K.J.;Hariharan,M.;Dewey,F.E.;Proteomics2005,5(13),3226−45.Cheng,Y.;Clark,M.J.;Im,H.;Habegger,L.;Balasubramanian,S.;(57)Banys-Paluchowski,M.;Witzel,I.;Riethdorf,S.;Rack,B.;Janni,O’Huallachain,M.;Dudley,J.T.;Hillenmeyer,S.;Haraksingh,R.;W.;Fasching,P.A.;Solomayer,E.F.;Aktas,B.;Kasimir-Bauer,S.;Sharon,D.;Euskirchen,G.;Lacroute,P.;Bettinger,K.;Boyle,A.P.;Pantel,K.;Fehm,T.;Muller,V.EvaluationofserumepidermalKasowski,M.;Grubert,F.;Seki,S.;Garcia,M.;Whirl-Carrillo,M.;growthfactorreceptor(EGFR)incorrelationtocirculatingtumorGallardo,M.;Blasco,M.A.;Greenberg,P.L.;Snyder,P.;Klein,T.E.;cellsinpatientswithmetastaticbreastcancer.Sci.Rep.2017,7(1),Altman,R.B.;Butte,A.J.;Ashley,E.A.;Gerstein,M.;Nadeau,K.C.;17307.Tang,H.;Snyder,M.Personalomicsprofilingrevealsdynamic(58)Brailo,V.;Vucicevic-Boras,V.;Lukac,J.;Biocina-Lukenda,D.;molecularandmedicalphenotypes.Cell2012,148(6),1293−307.Zilic-Alajbeg,I.;Milenovic,A.;Balija,M.Salivaryandserum(45)Greening,D.W.;Simpson,R.J.Acentrifugalultrafiltrationinterleukin1beta,interleukin6andtumornecrosisfactoralphainstrategyforisolatingthelow-molecularweight(

14JournalofProteomeResearchpubs.acs.org/jprArticleHalama,M.;Jenkins,G.;Sheppard,D.;Croner,R.S.;Christoph,J.;(78)Minami,S.;Sato,Y.;Matsumoto,T.;Kageyama,T.;Britzen-Laurent,N.;Naschberger,E.;Schellerer,V.;Sturzl,M.;Fried,Kawashima,Y.;Yoshio,K.;Ishii,J.;Matsumoto,K.;Nagashio,R.;M.;Rogler,G.;Scharl,M.beta6-integrinservesasanovelserumOkayasu,I.Proteomicstudyofserafrompatientswithbladdertumormarkerforcolorectalcarcinoma.Int.J.Cancer2019,145(3),cancer:usefulnessofS100A8andS100A9proteins.CancerGenomics678−685.Proteomics2010,7(4),181−189.(64)Larkin,S.E.;Johnston,H.E.;Jackson,T.R.;Jamieson,D.G.;(79)Lawicki,S.;Zajkowska,M.;Glazewska,E.K.;Bedkowska,G.E.;Roumeliotis,T.I.;Mockridge,C.I.;Michael,A.;Manousopoulou,A.;Szmitkowski,M.PlasmalevelsanddiagnosticutilityofVEGF,MMP-Papachristou,E.K.;Brown,M.D.;Clarke,N.W.;Pandha,H.;Aukim-9,andTIMP-1inthediagnosisofpatientswithbreastcancer.Hastie,C.L.;Cragg,M.S.;Garbis,S.D.;Townsend,P.A.DetectionOncoTargetsTher.2016,9,911−9.ofcandidatebiomarkersofprostatecancerprogressioninserum:a(80)Lee,J.H.;Choi,J.W.;Kim,Y.S.PlasmaorserumTIMP-1isadepletion-free3DLC/MSquantitativeproteomicspilotstudy.Br.J.predictorofsurvivaloutcomesincolorectalcancer:ameta-analysis.J.Cancer2016,115(9),1078−1086.Gastrointest.LiverDis.2011,20(3),287−291.(65)Kluger,H.M.;Hoyt,K.;Bacchiocchi,A.;Mayer,T.;Kirsch,J.;(81)Mroczko,B.;Lukaszewicz-Zajac,M.;Gryko,M.;Kedra,B.;Kluger,Y.;Sznol,M.;Ariyan,S.;Molinaro,A.;Halaban,R.PlasmaSzmitkowski,M.Clinicalsignificanceofserumlevelsofmatrixmarkersforidentifyingpatientswithmetastaticmelanoma.Clin.metalloproteinase2(MMP-2)anditstissueinhibitor(TIMP-2)inCancerRes.2011,17(8),2417−25.gastriccancer.FoliaHistochem.Cytobiol.2011,49(1),125−31.(66)Helgeland,E.;Breivik,L.E.;Vaudel,M.;Svendsen,O.S.;(82)Rieckmann,J.C.;Geiger,R.;Hornburg,D.;Wolf,T.;Kveler,Garberg,H.;Nordrehaug,J.E.;Berven,F.S.;Jonassen,A.K.K.;Jarrossay,D.;Sallusto,F.;Shen-Orr,S.S.;Lanzavecchia,A.;Mann,ExploringthehumanplasmaproteomeforhumoralmediatorsofM.;Meissner,F.Socialnetworkarchitectureofhumanimmunecellsremoteischemicpreconditioning–awordofcaution.PLoSOne2014,unveiledbyquantitative.Nat.Immunol.2017,18(5),583−593.9(10),e109279.(83)Agnoli,C.;Grioni,S.;Pala,V.;Allione,A.;Matullo,G.;(67)Dragutinovic,V.V.;Radonjic,N.V.;Petronijevic,N.D.;Tatic,Gaetano,C.D.;Tagliabue,G.;Sieri,S.;Krogh,V.BiomarkersofS.B.;Dimitrijevic,I.B.;Radovanovic,N.S.;Krivokapic,Z.V.Matrixinflammationandbreastcancerrisk:acase-controlstudynestedinthemetalloproteinase-2(MMP-2)and−9(MMP-9)inpreoperativeEPIC-Varesecohort.Sci.Rep.2017,7(1),12708.serumasindependentprognosticmarkersinpatientswithcolorectal(84)Chen,C.;Shen,H.;Zhang,L.G.;Liu,J.;Cao,X.G.;Yao,A.L.;cancer.Mol.Cell.Biochem.2011,355(1−2),173−8.Kang,S.S.;Gao,W.X.;Han,H.;Cao,F.H.;Li,Z.G.Construction(68)Patel,S.;Sumitra,G.;Koner,B.C.;Saxena,A.Roleofserumandanalysisofprotein-proteininteractionnetworksbasedonmatrixmetalloproteinase-2and−9topredictbreastcancerproteomicsdataofprostatecancer.Int.J.Mol.Med.2016,37(6),progression.Clin.Biochem.2011,44(10−11),869−72.1576−86.(69)Cymbaluk-Ploska,A.;Chudecka-Glaz,A.;Pius-Sadowska,E.;(85)Dahse,R.;Utting,M.;Werner,W.;Schimmel,B.;Claussen,U.;Machalinski,B.;Menkiszak,J.;Sompolska-Rzechula,A.SuitabilityJunker,K.TP53alterationsasapotentialdiagnosticmarkerinassessmentofbaselineconcentrationofMMP3,TIMP3,HE4andsuperficialbladdercarcinomaandinpatientsserum,plasmaandurineCA125intheserumofpatientswithovariancancer.J.OvarianRes.samples.Int.J.Oncol.2002,20(1),107−115.2018,11(1),1.(86)Nanjappa,V.;Thomas,J.K.;Marimuthu,A.;Muthusamy,B.;(70)Zhong,X.Y.;Kaul,S.;Bastert,G.EvaluationofMUC1andRadhakrishnan,A.;Sharma,R.;AhmadKhan,A.;Balakrishnan,L.;EGP40inbonemarrowandperipheralbloodasamarkerforoccultSahasrabuddhe,N.A.;Kumar,S.;Jhaveri,B.N.;Sheth,K.V.;Kumarbreastcancer.Arch.Gynecol.Obstet.2001,264(4),177−81.Khatana,R.;Shaw,P.G.;Srikanth,S.M.;Mathur,P.P.;Shankar,S.;(71)Braicu,C.;Tudoran,O.;Balacescu,L.;Catana,C.;Neagoe,E.;Nagaraja,D.;Christopher,R.;Mathivanan,S.;Raju,R.;Sirdeshmukh,Berindan-Neagoe,I.;Ionescu,C.ThesignificanceofPDGFR.;Chatterjee,A.;Simpson,R.J.;Harsha,H.C.;Pandey,A.;Prasad,expressioninserumofcolorectalcarcinomapatients–correlationT.S.PlasmaProteomeDatabaseasaresourceforproteomicswithDuke’sclassification.CanPDGFbecomeapotentialbiomarker?research:2014update.NucleicAcidsRes.2014,42(Databaseissue),Chirurgia(Bucur)2013,108(6),849−854.D959−D965.(72)Shevchenko,V.E.;Kovalev,S.V.;Arnotskaya,N.E.;(87)Adhikari,S.;Sharma,S.;Ahn,S.B.;Baker,M.S.InSilicoZborovskaya,I.B.;Akhmedov,B.B.;Polotskii,B.E.;Kostin,A.U.;PeptideRepertoireofHumanOlfactoryReceptorProteomesonMoukeria,A.F.;Zaridze,D.G.;Davidov,M.I.HumanbloodplasmaHigh-StringencyMassSpectrometry.J.ProteomeRes.2019,18(12),proteomemappingforsearchofpotentialmarkersofthelungsquamouscellcarcinoma.Eur.J.MassSpectrom.2013,19(2),123−4117−4123.(88)Schaeffer,M.;Gateau,A.;Teixeira,D.;Michel,P.A.;Zahn-33.(73)Lomholt,A.F.;Christensen,I.J.;Hoyer-Hansen,G.;Nielsen,Zabal,M.;Lane,L.TheneXtProtpeptideuniquenesschecker:atoolH.J.Prognosticvalueofintactandcleavedformsoftheurokinasefortheproteomicscommunity.Bioinformatics2017,33(21),3471−plasminogenactivatorreceptorinaretrospectivestudyof5183472.colorectalcancerpatients.ActaOncol.2010,49(6),805−11.(89)MacLean,B.;Tomazela,D.M.;Shulman,N.;Chambers,M.;(74)Almasi,C.E.;Hoyer-Hansen,G.;Christensen,I.J.;Pappot,H.Finney,G.L.;Frewen,B.;Kern,R.;Tabb,D.L.;Liebler,D.C.;PrognosticsignificanceofurokinaseplasminogenactivatorreceptorMacCoss,M.J.Skyline:anopensourcedocumenteditorforcreatinganditscleavedformsinbloodfrompatientswithnon-smallcelllungandanalyzingtargetedproteomicsexperiments.Bioinformatics2010,cancer.APMIS2009,117(10),755−61.26(7),966−968.(75)Kjellman,A.;Akre,O.;Gustafsson,O.;Hoyer-Hansen,G.;(90)Reiter,L.;Rinner,O.;Picotti,P.;Huttenhain,R.;Beck,M.;Lilja,H.;Norming,U.;Piironen,T.;Tornblom,M.SolubleurokinaseBrusniak,M.Y.;Hengartner,M.O.;Aebersold,R.mProphet:plasminogenactivatorreceptorasaprognosticmarkerinmenautomateddataprocessingandstatisticalvalidationforlarge-scaleparticipatinginprostatecancerscreening.J.Intern.Med.2011,269SRMexperiments.Nat.Methods2011,8(5),430−5.(3),299−305.(91)Govaert,E.;VanSteendam,K.;Willems,S.;Vossaert,L.;(76)Cheng,Y.;Liu,C.;Zhang,N.;Wang,S.;Zhang,Z.ProteomicsDhaenens,M.;Deforce,D.Comparisonoffractionationproteomicsanalysisforfindingserummarkersofovariancancer.BiomedRes.Int.forlocalSWATHlibrarybuilding.Proteomics2017,17,1700052.2014,2014,179040.(92)Navarro,P.;Kuharev,J.;Gillet,L.C.;Bernhardt,O.M.;(77)Schmitz,M.;Grignard,G.;Margue,C.;Dippel,W.;Capesius,MacLean,B.;Röst,H.L.;Tate,S.A.;Tsou,C.C.;Reiter,L.;Distler,C.;Mossong,J.;Nathan,M.;Giacchi,S.;Scheiden,R.;Kieffer,N.U.;Rosenberger,G.;Perez-Riverol,Y.;Nesvizhskii,A.I.;Aebersold,CompletelossofPTENexpressionasapossibleearlyprognosticR.;Tenzer,S.Amulticenterstudybenchmarkssoftwaretoolsformarkerforprostatecancermetastasis.Int.J.Cancer2007,120(6),label-freeproteomequantification.Nat.Biotechnol.2016,34(11),1284−92.1130−1136.2388https://doi.org/10.1021/acs.jproteome.0c00898J.ProteomeRes.2021,20,2374−2389

15JournalofProteomeResearchpubs.acs.org/jprArticle(93)Tully,B.;Balleine,R.L.;Hains,P.G.;Zhong,Q.;Reddel,R.R.;Robinson,P.J.AddressingtheChallengesofHigh-ThroughputCancerTissueProteomicsforClinicalApplication:ProCan.Proteomics2019,19,1900109.(94)Aggarwal,S.;Yadav,A.K.FalseDiscoveryRateEstimationinProteomics.MethodsMol.Biol.2016,1362,119−28.(95)Buscail,E.;Alix-Panabieres,C.;Quincy,P.;Cauvin,T.;̀Chauvet,A.;Degrandi,O.;Caumont,C.;Verdon,S.;Lamrissi,I.;Moranvillier,I.;etal.HighClinicalValueofLiquidBiopsytoDetectCirculatingTumorCellsandTumorExosomesinPancreaticDuctalAdenocarcinomaPatientsEligibleforUp-FrontSurgery.Cancers2019,11(11),1656.(96)Voskuil,J.Commercialantibodiesandtheirvalidation.F1000Research2014,3,232−232.(97)Anderson,N.L.;Anderson,N.G.Thehumanplasmaproteome:history,character,anddiagnosticprospects.Mol.CellProteomics2002,1(11),845−67.(98)Doerr,A.DIAmassspectrometry.Nat.Methods2015,12(1),35−35.(99)Zhang,F.;Ge,W.;Ruan,G.;Cai,X.;Guo,T.Data-IndependentAcquisitionMassSpectrometry-BasedProteomicsandSoftwareTools:AGlimpsein2020.Proteomics2020,20,1900276.(100)Doellinger,J.;Blumenscheit,C.;Schneider,A.;Lasch,P.IsolationWindowOptimizationofData-IndependentAcquisitionUsingPredictedLibrariesforDeepandAccurateProteomeProfiling.Anal.Chem.2020,92(18),12185−12192.(101)Shen,J.;Pagala,V.R.;Breuer,A.M.;Peng,J.;Bin,M.;Wang,X.SpectralLibrarySearchImprovesAssignmentofTMTLabeledMS/MSSpectra.J.ProteomeRes.2018,17(9),3325−3331.(102)Rice,S.J.;Liu,X.;Zhang,J.;Belani,C.P.AbsoluteQuantificationofAllIdentifiedPlasmaProteinsfromSWATHDataforBiomarkerDiscovery.Proteomics2019,19,1800135.(103)Rosenberger,G.;Liu,Y.;Röst,H.L.;Ludwig,C.;Buil,A.;Bensimon,A.;Soste,M.;Spector,T.D.;Dermitzakis,E.T.;Collins,B.C.;Malmström,L.;Aebersold,R.InferenceandquantificationofpeptidoformsinlargesamplecohortsbySWATH-MS.Nat.Biotechnol.2017,35(8),781−788.(104)Collins,B.C.;Hunter,C.L.;Liu,Y.;Schilling,B.;Rosenberger,G.;Bader,S.L.;Chan,D.W.;Gibson,B.W.;Gingras,A.C.;Held,J.M.;Hirayama-Kurogi,M.;Hou,G.;Krisp,C.;Larsen,B.;Lin,L.;Liu,S.;Molloy,M.P.;Moritz,R.L.;Ohtsuki,S.;Schlapbach,R.;Selevsek,N.;Thomas,S.N.;Tzeng,S.C.;Zhang,H.;Aebersold,R.Multi-laboratoryassessmentofreproducibility,qualitativeandquantitativeperformanceofSWATH-massspectrom-etry.Nat.Commun.2017,8(1),291.(105)Ahn,S.B.;Mohamedali,A.;Anand,S.;Cheruku,H.R.;Birch,D.;Sowmya,G.;Cantor,D.;Ranganathan,S.;Inglis,D.W.;Frank,R.;Agrez,M.;Nice,E.C.;Baker,M.S.Characterizationoftheinteractionbetweenheterodimericαvβ6integrinandurokinaseplasminogenactivatorreceptor(uPAR)usingfunctionalproteomics.J.ProteomeRes.2014,13(12),5956−64.(106)Guo,H.;Ling,C.;Ma,Y.Y.;Zhou,L.X.;Zhao,L.Prognosticroleofurokinaseplasminogenactivatorreceptoringastricandcolorectalcancer:Asystematicreviewandmeta-analysis.Oncol.TargetsTher.2015,8,1503−1509.(107)Pizzatti,L.;Panis,C.;Lemos,G.;Rocha,M.;Cecchini,R.;Souza,G.H.;Abdelhay,E.Label-freeMSEproteomicanalysisofchronicmyeloidleukemiabonemarrowplasma:disclosingnewinsightsfromtherapyresistance.Proteomics2012,12(17),2618−31.2389https://doi.org/10.1021/acs.jproteome.0c00898J.ProteomeRes.2021,20,2374−2389