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

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

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