Comprehensive Analysis of Tryptic Peptides Arising from Disul fi de Linkages in NISTmAb and Their Use for Developing a Mass Spectral Lib

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pubs.acs.org/jprArticleComprehensiveAnalysisofTrypticPeptidesArisingfromDisulfideLinkagesinNISTmAbandTheirUseforDevelopingaMassSpectralLibraryQianDong,*XinjianYan,YuxueLiang,SanfordP.Markey,SergeyL.Sheetlin,ConcepcionA.Remoroza,WilliamE.Wallace,andStephenE.SteinCiteThis:J.ProteomeRes.2021,20,1612−1629ReadOnlineACCESSMetrics&MoreArticleRecommendations*sıSupportingInformationABSTRACT:Thisworkpresentsmethodsforidentifyingandthencreatingamassspectrallibraryfordisulfide-linkedpeptidesoriginatingfromtheNISTmAb,areferencematerialofthehumanizedIgG1kmonoclonalantibody(RM8671).Analysesinvolvedbothpartiallyreducedandnon-reducedsamplesunderneutralandweaklybasicconditionsfollowedbynanoflowliquidchromatographytandemmassspectrometry(LC−MS/MS).SpectraofpeptidescontainingdisulfidebondsareidentifiedbybothMS1ionandMS2fragmentiondatainordertocompletelymapallthedisulfidelinkagesintheNISTmAb.Thisledtothedetectionof383distinctdisulfide-linkedpeptideions,arisingfromfullytrypticcleavage,missedcleavage,irregularcleavage,complexMet/Trpoxidationmixtures,andmetaladducts.Fragmentationfeaturesofdisulfidebondsunderlow-energycollisiondissociationwereexamined.Theseinclude(1)peptidebondcleavageleavingdisulfidebondsintact;(2)disulfidebondcleavage,oftenleadingtoextensivefragmentation;and(3)doublecleavageproductsresultingfrombreakagesoftwopeptidebondsorbothpeptideanddisulfidebonds.AutomatedannotationofvariouscomplexMS/MSfragmentsenabledtheidentificationofdisulfide-linkedpeptideswithhighconfidence.Peptidescontainingeachoftheninenativedisulfidebondswereidentifiedalongwith86additionaldisulfideDownloadedvia157.33.32.30onMay14,2021at07:54:19(UTC).linkagesarisingfromdisulfidebondshuffling.Thepresenceofshuffleddisulfideswasnearlycompletelyabrogatedbyrefiningdigestconditions.Acuratedspectrallibraryof702disulfide-linkedpeptidespectrawascreatedfromthisanalysisandispubliclyavailableforfreedownload.SinceallIgG1antibodieshavethesameconstantregions,theresultinglibrarycanbeusedasatoolforfacileidentificationof“hard-to-find”disulfide-bondedpeptides.Moreover,weshowthatonemayidentifysuchpeptidesoriginatingfromIgG1proteinsinhumanserum,therebyservingasameansofmonitoringthecompletenessofproteinreductioninproteomicsSeehttps://pubs.acs.org/sharingguidelinesforoptionsonhowtolegitimatelysharepublishedarticles.studies.DataareavailableviaProteomeXchangewithidentifierPXD023358.KEYWORDS:cysteine,disulfide,disulfide-linkedpeptides,NISTmAb,IgG1,MSspectrallibrary,fullMSscan,disulfidebondfragmentation■INTRODUCTIONalkylationtopreventthedisulfidebondfromre-forming.TheseDisulfidebridgesbetweencysteines(Cys)stabilizethethree-well-establishedproteindigestionmethodscanfullysequencedimensionalstructureofproteins,aidingwithproteinfoldingindividualproteinsandidentifyalargefractionoflow-andultimatelyenablingproteinfunctionality.1,2Dysregulatedabundancemodificationsinproteindigests.Recently,twostructuraldisulfidebondsarecharacteristicofcertainhumandiseases,suchasvariousneurodegenerativediseasesandReceived:October16,20203cancers.Therefore,detailedinformationondisulfidecon-Published:February8,2021nectivityiscriticalforproteinstructure−functionstudiesandbiotherapeuticsstructureintegrityassessment.Typicalproteo-micsexperimentsdissociatedisulfidebondsbyreductionaspartofthedenaturingprocessandthencapCysresiduesbyNotsubjecttoU.S.Copyright.Published2021byAmericanChemicalSocietyhttps://dx.doi.org/10.1021/acs.jproteome.0c008231612J.ProteomeRes.2021,20,1612−1629

1JournalofProteomeResearchpubs.acs.org/jprArticledetailedmassspectrallibrary-basedexaminationsofthe■MATERIALSANDMETHODSNISTmAb,areferencematerialofthehumanizedIgG1κmonoclonalantibody(RM8671),4anditsglycosylationwereMaterials5,6reported.Incontrast,MSanalysisofdisulfidebondsrequiresTheprimarysample8670NISTmAb(PS8670,anin-houseadifferentanalyticalapproach.Recentadvancesinmassprimarystandardmaterial),lot3f1b,isanIgG1kmAbderivedspectrometryhaveenabledthedevelopmentofcustomizedfromaseparateproductionlotofNISTmAb8671expressedin7−94LC−MSstrategiesfordisulfidebondmappinginproteins.murinesuspensionculture.ItwasobtainedfromtheAmajorrequirementinsamplepreparationistopreventnon-BioanalyticalScienceGroupatNIST.Digestionreagentsnativedisulfideformation(i.e.,disulfidebondartifacts).Theguanidinehydrochloride,dithiothreitol(DTT),tris(2-disulfideshufflingoccursspontaneouslyinthepresenceoffreecarboxyethyl)phosphine(TCEP),N-ethylmaleimide(NEM),cysteinesatpH7.5−8.5,whichistypicalforproteindigestionandiodoacetamide(IAM)werepurchasedfromSigma-Aldrichinstandardproteomicsprotocols.10,11Theseartifactscanbe(St.Louis,MO).ArevisedTrisbuffer(pH=7)waspreparedminimizedbyblockingfreecysteinesandusingacidictoviaadjustmentoftrisbasesolution(200mmol)byHClneutralpHconditions.Methodsfordeterminingdisulfidesolution(1mol/L).Sequencing-gradetrypsinwaspurchasedpatternsincludeliquidchromatographicultravioletprofilefromPromega(Madison,WI).RapiGest,brand-nameforcomparisonbetweenreducedandnon-reducedpeptides,directsodium3-[(2methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate,waspurchasedfromWaters(Milford,massspectrometrydetectionofdisulfidewithoutreductionorMA);Zebaspincolumns(7Kmolecularweightcutoffpartialreduction,chemicallabelingidentification,andtandem12−17(MWCO))werepurchasedfromThermoFisherScientificmassspectrometrysequencingofnon-reducedpeptides.(Waltham,MA).Chromatographicseparationswereper-SignificantchallengesremainindisulfideanalysesduetoformedonanAcclaimpepmap100nanocolumn(150mm×complexintertwineddisulfides,disulfideshuffling,incomplete75μm,C18,3μmparticlesize,100Åporesize,Dionex,digestionofnon-reducedproteins,largepeptidemasses,and18−24Sunnyvale,CA).lowionizationefficiencyofdisulfide-linkedpeptides.TandemmassspectraofpeptidescontainingdisulfidebondsTrypticDigestionaregenerallyverycomplexbecauseofvariouscollisionalTogenerateawidevarietyoftrypticpeptidesthatweredissociationproductsarisingfromcleavageofthecomponentcollectedintheNISTmAbpeptidespectrallibrary,parameterspeptidebackboneaswellasdisulfidebonds.Therearenoforaseriesofdigestionprotocolvariationswereusedduringdiagnosticfragmentscharacteristicofdisulfidespectraunlikedenaturing,reduction,andalkylationoftheNISTmAb.The5otherpost-translationalmodificationsthatarereadilydetecteddetailsaredescribedinourpreviouspaper.Amongthem,thebypredominantionsresultingfromresiduesidechaincleavagetwoprotocolsofthenon-reducedandpartiallyreduced(e.g.,oxoniumionsofglycans,phosphoricacid(H3PO4)samplespreparedunderthesegeneralexperimentalconditionsneutrallossofO-phosphorylation).Thus,mostavailable(pH=8)wereusedinthisstudy,therefore,disulfidebondbioinformaticstoolscurrentlyareincapableofrecognizingrearrangementscouldoccur.Topreventtheseshufflingvariousdisulfide-linkedionsandinterpretingtheirproductionreactions,twoalternativemethodsareusedforsamplespectra.preparationatpH7.AllmethodsusedinanalyzingdisulfidesThepresentstudyisacontinuationofourpreviouswork,5,6aredescribedbelow.inwhichtwocomprehensivespectrallibrariesweredevelopedGeneralExperimentalConditions(pH=8).NISTmAbforcharacterizingpeptides,glycopeptides,andotherbio-(500μg)wasdenaturedatroomtemperaturefor10minusinglogicallymodifiedpeptidesderivedfromtheNISTmAb.Theseguanidinehydrochloride(6M)in50μLof100mmol/LTrislibrariescontainalargenumberoflow-abundancemodifica-HCI(Tris(hydroxymethyl)aminomethanehydrochloride)buf-fer(pH=8).Reductionandalkylationwerevariedasfollows:tionsoccurringonawiderangeofresidues.Notably,many(a)thesamplewasneitherreducednoralkylatedor(b)themodificationsarenotreportedbymostcommonlyusedsamplewaspartiallyreducedbyadding5μLof200mmol/LpeptideidentificationprogramssincemodifiedpeptidesdonotTCEPtotheabovedenaturedmixtureandincubatingatroomfragmentprimarilybypeptidebackbonecleavages.Instead,temperaturefor1h,followedbyalkylationwith10μLof200theirsidechaincleavagereactionscandominatetheirproductmmol/LIAMatroomtemperatureinthedarkfor1h.Theionspectra.ThisstudyextendsthepriorworktoenablethealkylatedproteinsolutionwasdesaltedwithaZebaspinidentificationofanotherclassofhard-to-findpeptideproductscolumn(7KMWCO)priortoadding10μgofPromegaofdigestion,namely,disulfide-bondedpeptides.Toidentifytrypsin.Theresultingmixturewassubjectedtodigestionwithandconfirmtheidentityofthesecomplexpeptides,datasetssequencing-gradetrypsinat37°Cfor0.25,2,and18h.generatedbytwodigestionprotocols,non-reductionandNon-reductionandAlkylationwithNEM(pH=7).500partialreduction,werechosenfromalargeamountofdataμgofNISTmAbwasdenaturedatroomtemperaturefor10obtainedbyaseriesofcommonprotocolsusedincreatingtheminusing6Mguanidinein50μLoftrisbuffer(pH=7).The5abovespectrallibraries.ThedataanalysismethoddevelopedalkylationreagentNEMwasusedbecauseofitsthiolspecificityinthisworkemploysfullscanMSspectratoidentifypotentialandstabilityatpH7,contrastingtoIAMwithoptimalreactionprecursorionsandvalidatesthembyadetailedfragmentationconditionsatpH8,whichmostlikelyresultsindisulfideanalysisoftheirtandemmassspectra(MS/MSspectra).shuffling.810μLof200mmol/LNEMwasaddedtotheaboveResultingspectrathenservetocreateasearchablemassdenaturedmixtureandincubatingatroomtemperatureinthespectrallibrary.Asabonus,wefindanddemonstratetheutilitydarkfor1h.Thealkylatedproteinsolutionwasdesaltedasofthesespectraforidentifyingdigested,butincompletelydescribedabove.Trypsin(10μg)wasaddedtodigestthereducedIgG1-derivedpeptidesinhumanserum,therebysolutionat37°Cfor0.25,2,and18h.Thereactionwasprovidingauniquemeasureofreductioneffectiveness.quenchedwith5μLofformicacid(50%v/v).1613https://dx.doi.org/10.1021/acs.jproteome.0c00823J.ProteomeRes.2021,20,1612−1629

2JournalofProteomeResearchpubs.acs.org/jprArticleTable1.ClassificationoftheThreeMajorGroupsofProductIonsObservedintheSpectraofDisulfidePeptideDiscussedinThisSection,SGTASVVCLLNNFYPR_SS_VYACEVTHQGLSSPVTKaaSymbols|,-H2S,and+S,-2Hand+0denotecleavageofamide,C−S,andS−Sbonds,respectively.ReductionwithTCEPandAlkylationwithNEM(pH=abundance,andretentiontimes(RT).Thissetofpeaksis7).500μgofNISTmAbwasdenaturedusing6MofdenotedasanionclusterbyProMS.ProMSidentifiesisotopesguanidine;reductionwasperformedbyadding5μLof200ofeachionclustertodetermineitschargestate,retentiontime,mmol/LTCEPtotheabovedenaturedmixtureandincubatingabundance,andmonoisotopicm/zinallLC−MS/MSruns.atroomtemperaturefor1h,followedbyalkylationwith20μLTheabundanceofanionclusteriscalculatedusingthepeakof200mmol/LNEMatroomtemperatureinthedarkfor1h.areaofallobservableisotopepeaks.TheRTofanionclusterThen,thealkylatedproteinsolutionwasdesaltedasdescribedreferstoatimewhentheaccumulatedabundanceisthehalfofabove.Trypsin(10μg)wasaddedtodigesttheproteinthetotalabundances.Additionally,theRTrangeofapeptideissolutionat37°Cfor0.25,2,and18h.Thereactionwasdefinedasthefirstandlastelutingvaluesofthepeptide.quenchedwith5μLofformicacid(50%v/v).EstimationoftheMedianRTRange.TheRPLCcolumnLC−MS/MSAnalysiscangenerateconsistentpeptideRTundersameexperimentalconditions,withsmallvariationsfromruntorun.Inthisstudy,Theabovedigests(0.2μg)wereanalyzedonaDionexthemedianvalueofRTrangeforspecificexperimentsisusedUltimate3000RSLCNanoLCwithanAcclaimpepmap100inadiscriminantfiltertoremoveunrelatedidentifications.ThecolumnwithananospraysourceconnectedtooneoftwomassfirststepistoextracttheRTrangeofthemostabundantionofspectrometers:aQExactiveHybridQuadrupole-Orbitrapeachpeptide;thenthemedianRTrangeiscalculatedfrommassspectrometeroranOrbitrapFusionLumosmassreplicaterunsunderidenticalexperimentconditions.spectrometer(ThermoFisherScientific,Waltham,MA)inDisulfidePeptideAnalysis.TheIgG1disulfidebondthepositiveionmode.MobilephaseAconsistedof0.1%structure(seeFigureS1)ishighlyconservedwithasimpleformicacidinwater,andmobilephaseBconsistedof0.1%disulfideconnectivitypatternbecausemostdisulfidebondsareformicacidinACN.Thepeptideswereelutedbyincreasingconstrainedwithinanindividualheavy/lightdomain.InallbutmobilephaseBfrom1%to90%over120min.Datawasonecase,componentpeptidescanbefoundtobeconnectedcollectedusingadata-dependentmodewithadynamicbyasinglenativedisulfidebond.Theexceptioninvolvestheexclusionof20s.Thetop10mostabundantprecursorionsidenticalpeptidesfromtwoheavychainslinkingtoeachotherwereselectedfroma250m/zto1850m/zfullscanforHCDinthehingeregionbytwodisulfidebonds(HC229-HC229andiontrapfragmentation(FT-CID)withnormalizedandHC232-HC232).Morecomplexdisulfideconnectivitycollisionalenergy(NCE)parameters(NCEof16,20,24,patternsinvolvingmultiple,intertwineddisulfidebondsand32,and36).TheresolutionoffullMSscanandMS/MSscantheproximityofCysresiduesinapeptidesequencerequirewassetat120,000and30,000ontheFusionLumosandatmanualinvestigationoralternativeMSfragmentation70,000and17,500ontheQExactive,respectively.techniques.Theseareoutsidethescopeofthispaper.DataAnalysisSpectraofDisulfide-LinkedPeptides.Eachdisulfide-MS1analysisofAllDetectableIonClustersinaLC−bondedpeptideinthisstudyiscomposedoftwocomponentMSRun.AlldetectableMS1ionsinaLC−MSrunwerepeptides:peptide1(P1)andpeptide2(P2).Inthiswork,25analyzedwithanin-houseprogram,NISTProMS.InaLC−whentheyareinseparatepolypeptidechains(heavy,H,orMSpeptidemappinganalysis,eachpeptideioncanbedetectedlight,L),theonefromtheheavychainisshownfirst.WheninviaasetofMS1(LC−MS)peakswithspecificisotopes,thesamechain,theyareshowninorderofsequencenumber.1614https://dx.doi.org/10.1021/acs.jproteome.0c00823J.ProteomeRes.2021,20,1612−1629

3JournalofProteomeResearchpubs.acs.org/jprArticleTheexamplegivenbelowisapeptideconnectedbytwo■RESULTSconsecutiveCysresiduesintheconstantregionoftheLchain.IdentificationofDisulfideBondsItisrepresentedintwowaysinthispaper:DataAnalysisStrategy.Disulfide-linkedpeptidescannotbeidentifiedbymostcurrentdatabasesearchingtoolssince7−9theyassumelinearpeptides.Inrecentdisulfidestudies,the(2)SGTASVVCLLNNFYPR_SS_VYA-anticipatedpolypeptidescontainingdisulfidelinkagesareoftenCEVTHQGLSSPVTK.usedforinitialsearchingofMS2spectraacquiredfromnon-Forassigningproductionsofdisulfide-bondedpeptides,reducedorpartiallyreduceddigests,andthespectramatchedbytheoreticalmassarethenconfirmedbyproductioneachspectrumcanbeconsideredasamixtureproducedfromassignment.However,becauseofthecomplexityofdisulfideP1modifiedbyP2attheCysresidueandP2modifiedbyP1atbondfragmentation,manyofsuchpeptidesmaybeundetectedtheCysresidue.Forexampleormisidentified.Toaddressthisissue,weusedapreviouslyMS/MSspectrum=bandyionseriesfrom“SGTASVVC-developedalternativeworkflowforcharacterizinghard-to-(P2-2H)LLNNFYPR”+b(2)andy(2)ionseriesfromidentifypeptidesandmodificationsderivedfromthe“VYAC(P1-2H)EVTHQGLSSPVTK”5NISTmAbinanLC−MS/MSanalysis.OurmethodstartsAsshownintheexpression,inordertodistinguishfragmentswithsearchingforMS1ionclusterstofindalldisulfide-fromdifferentcomponentsindisulfide-containingspectra,ionscontainingcandidatesofinterestbasedontheproteinfromP2arelabeledby“(2)”followingionseriesofa,b,ory.sequenceandthenassignstheirproductionspectratotheinClassificationofProductIons.Mostoftheproductionssilicofragmentationofspecificdisulfide-linkedpeptidesforofdisulfidebondfragmentationobservedinthisworkfallintovalidatingidentificationresults.threemaingroups,asshowninTable1.Figure1isaflowchartoftheprocessofidentifyingspectraofGroup1:Fragmentswithanintactdisulfidebond.Theyaredisulfide-linkedpeptides,whicharesubsequentlyusedforfurtherdividedintothreesubgroups.IonsresultingfromsinglepeptidebackbonecleavageinP1orP2arelabeledusingthestandardnotationofa/a(2),b/b(2),andy/y(2)ionseries,showninSubgroup1aofTable1.IonsarisingfromdoublecleavageinP1orP2aredenotedasinternalions,suchas“intVCLL”showninSubgroup1b.Ifafragmentsequencecontainsmorethanfourresidues,thepositionrangeisused,suchasint7_12.Whenionsarecomposedoftwofragments(P1andP2fragments),theyaredenotedbybothproductions,suchas“[y10:b(2)5]”inSubgroup1c.Group2:Specificfragmentionsinvolvingacleavageofdisulfidebonds,i.e.,carbon−sulfur(C−S)andsulfur−sulfur(S−S).C−Sbondcleavageyieldsfragments(Subgroup2a)withamodificationofpersulfide(+S,31.9721Da)ordehydroalanine(-H2S,−33.9877Da)atCys,whileS−Sbondcleavagegeneratesfragments(Subgroup2b)withamodificationofCys(+0Da)andCysthioaldehyde(-2H,−2.0156Da).Group3:IonsareduetosingleordoublepeptidebackboneFigure1.Buildinglibraryofreferencemassspectrafordisulfidecleavagesbutdonotcontainadisulfidebond,soaanalysis.conventionalannotationisused(Subgroups3aand3b).ProductIonAssignment.AsoftwaretoolwasdevelopedformatchingexperimentalMS/MSfragmentionsfromHCDcreatingaspectrallibrary.First,theoreticalm/zvaluesareandFT-CIDspectratotheinsilicofragmentationofthecomputedforallpotentialdisulfide-linkedpeptidesderivedcomponentpeptidesaswellasdisulfidebonds.First,anfromknownnativeandpotentiallyscrambleddisulfidebondanalysisoffragmentisotopicdistributionswasperformed,linkagesbasedonproteinsequence(step1).Second,allwhichallowstheaccuratemassdeterminationofproductiondetectableMS1peaksareanalyzedtodeterminetheirmonoisotopicpeaksinMS/MSspectra.Thedeviationmonoisotopicm/z,charge,abundance,andRTofeachionthresholdtoacceptanisotopicassignmentis20ppm.Forcluster(step2).Inthethirdstep,theinitialidentificationisannotatingfragments,theprogrammakesassignmentsintheconductedbymatchingobservedm/zvaluesderivedfromsteporderoftheoccurrencefrequencyoffragmentions:(1)neutral2withtheprecursorm/zvaluesinstep1.ThemedianRToflossofprecursorions;(2)amidebondcleavageionserieswiththesematchedionsinthesamedigestionconditionsisandwithoutadisulfidebondandtheircommonneutrallossescalculatedandusedtofilteridentificationsofionswith(H2O,NH3,NH3H2O,or2H2O);(3)disulfidecleavedatC−SinconsistentRT.Afterdisulfide-linkedpeptidesaretentativelyandS−Sbonds;(4)doublecleavagewithandwithoutaidentified,step4extractsMS2spectraandconductsfragmentdisulfidebond;and(5)neutrallosses(C2H4OandC2H6O2)ionanalysisbyinterpretingtheirproductionspectra.Instep5,frompeptidebondcleavageionseries.Allfragmentsthathavereplicatespectraareselectedbasedonproductionassign-reasonableisotopepatternsareanalyzed,andthosewithments.Theselectedspectrumshouldincludeatleast60%ofdeviationwithin20ppmbetweenobservedandtheoreticalm/peakabundancesthatcanbeassignedtofragmentsofdisulfide-zvaluesareannotated.specificstructureandpeptidesequence.Lastly,allvalidated1615https://dx.doi.org/10.1021/acs.jproteome.0c00823J.ProteomeRes.2021,20,1612−1629

4JournalofProteomeResearchpubs.acs.org/jprArticle101009090908090811090809090808080711090908080808081007101010101010101010101010101010101010101010101010101010×××××××××××××××××××××××××××absoluteabund.MS/MSAnalysesofthe18hNISTmAbDigest−VTNMDPADTATYYC8721.1635,6,7,8VTNMDPADTATYYC3.29888.7927,88.3_SS__SS_VTNMDPADTATYYCARDVTNMDPADTATYYCARDMI8213.8825,6,79381.5116,7DTSKNQVVLKVTNMDPADT9.39326.5018.66,7,82.3_SS__SS__SS_VTNMDPADTATYYCAR4286.02233.4VTNMDPADTATYYCAR4085.9064,52.4_SS_VVSVLTVLHQDWLNGKEYKCKVVSVLTVLHQDWLNGKEYKCKVSNKEYKCKCKVSNK4965.5264537.2875,64,5,6,76.61.5_SS__SS__SS__SS__SS_CKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV8259.11SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT6,7TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ6869.4285,6,77069.544SLSSVVTVPSSSLGTQTYICNVNHKPSNTK4.86SVVTVPSSSLGTQTYICNVNHKPSNTKSSVVTVPSSSLGTQTYICNVNHKPSNTK4.5TVPSSSLGTQTYICNVNHKPSNTK1.84409.2215,64122.0734209.1054,5,64,53.33836.9042.24,51.49.3CK_SS__SS__SS__SS__SS__SS__SS__SS__SS_CKVFSCSVMHEALHNHYTQKdeLinkagesObtainedfromtheTriplicateLCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS7898.9095,64.4fiC_SS__SS_NVNHKPSNTKCESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIR_SS_VTNMDPADTATYYCARESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIR5431.5874,5,63.6STSGGTAALGCLVK_SS_DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVSTSGGTAALG7916.9195,6,7,81.8TPEVTCVVVDVSHEDPEVK_SS_CKNQVSLTCLVK_SS_WQQGNVFSCSVMHEALHNHYTQK2328.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,sitespeptidesequencemasschargegmissedcleavedtryptic25%missedcleav.01semi-tryptictryptic94%0missedcleav.ESGPALVKPTQTLTLTCTFSGF01semi-tryptic0STSGGTAALGCLVKtryptic96%missedcleav.02semi-trypticTPEVTCVVVDVSHEDPEVK0tryptic99%semi-trypticTPEVTCVVVDVSHED00NQVSLTCLVKde-LinkedPeptidesDetectedforEachoftheNineNativeDisulfiHC22-HC971HC147-HC2032HC264-HC3243HC370-HC428HHHHVCCCbondnameapositioninproteinclassaaaTable2.DisulPartiallyReducedwithTCEPandAlkylatedwithIAM12341616https://dx.doi.org/10.1021/acs.jproteome.0c00823J.ProteomeRes.2021,20,1612−1629

5JournalofProteomeResearchpubs.acs.org/jprArticle100908111008071009090810100908hIn101010101010101010101010101010×××××××××××××××absoluteabund.Notes:Bond:gListheinterchainbond−HcfIntrapeptideform.5889.9625,6,7,8,96323.1254,5,6,7,8,91.22.2idesequence:sequenceoftwocomponenthetotalpeptideabundanceofthelinkagesite.NoteSFNRGEC4788.2994,5,62.7_SS_GECSFNRGEC3971.8833467.6394,5,6,73,4,5,6,73.32.6(CAM)_SS__SS_SC_2(SS)_(CAM)FSGSGSGTEFTLTISSLQPDDFATYYCFQ7320.34451.0SCTHTCPPCPAPELLGGPSVF4649.29855.4_SS__2(SS)__2(SS)_representthevariableandconstantdomainsoftheLchain.LOxidizedand/ormetallatedformswerealsodetected.ePAPELLGGPSVFLFPPKPKPAPELLGGPSVFLFPPKPKPAPELLGGPSVFLFPPKPKDTLMISRHKVYACEVTHQGLSSPVTKADYEKHKVYACEVTHQGLSSPVTKVYACEVTH4427.1683820.9035,63,4,5,6,76.26.0andC2658.25744.5LV_SS__SS__SS_(CAM)(CAM)(CAM)bde-linkedpeptide.Absoluteabund.:summedabsoluteabundanceoverallchargestatesofapeptide.fiPPCPPCPPCFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKVEIKR6071.8515,62.0_SS_(CAM)(CAM)(CAM)SFNRGEC1260.4862,3,47.8DKTHTCPPCPAPELLGGPSVFLFPPKPK_SS_(CAM)DKTHTCPPCPAPELLGGPSVFLFPPKPKDKTHTCPPCPAPELLGGPSVFLFPPKPKGSGYPFTFGGGTKedwithcarbamidomethylated(CAM)cysteineresidues.fidebondsconnectingthetwoHchains.2VTITCSASSR2SGTASVVCLLNNFYPR231SCDKTHTCSCDKTHTCCSCDK2Sfisitespeptidesequencemasschargemissedcleavedde-linkedpeptide.z:chargestateofdisulfitryptic92%missedcleav.01tryptic66%missedcleav.VTITCSASSR_SS_FSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKDIQMTQSPSTLSASVGDRVTITCSASSRsemi-tryptic015446.464,5SGTASVVCLLNNFYPR_SS_VYACEVTHQGLSSPVTK0SGTASVVCLLNNFYPRSGTASVVCLLNNFYPR1.4tryptic82%missedcleav.01semi-trypticTHTCPPCPAPELLGGPSVFLFPPKPK_2(SS)_THTCPPCPAPELLGGPSVFLFPPKPKTHTCPPCPAPELLGGPSVFLFPPKPK3555.74905456.7993,4,54,5,6,7,8THTCPPCPAPELLGGPSVFLFPPKPK6.71.2debonds7,8,and9wereidentifiHingeregioncontainsthetwodisulddebond.Mass:massofdisulfi3refertothevariableandconstantdomains1to3inHchain,respectively.HHC232LC23-LC87LC133-LC193HC229-HC229andHC232-2,andCH1,CdHdebondnamedbydomains.Position:Cyspositionineachproteinchain.Class:peptideclass.Misscleav.:totalnumberofmissedcleavagesites.PeptLL,CfiVCH-LHC223-LC213Hingemissedcleav.H1SCDKTHTCbondnameb5positioninproteinb6classc78and9VTable2.continuedalinkingtheHandLchains.disulpeptideslinkedbyadisulColumn3,thefullytrypticpeptideisshowninbold,andtherelativeabundanceiscalculatedasthepercentageofthetrypticpeptideabundanceovertthatseveralpeptidescontainingdisul1617https://dx.doi.org/10.1021/acs.jproteome.0c00823J.ProteomeRes.2021,20,1612−1629

6JournalofProteomeResearchpubs.acs.org/jprArticlereplicatespectraaremergedtoformconsensusspectraforresiduesaredetectedinthecompositespectrumgeneratedinclusioninaspectrallibrary(step6).frommultiplemono-/dioxidizedproducts.DisulfidepeptidesIdentificationofNative(Expected)NISTmAbDisul-originatedfromirregularcleavage(semitryptic)wereidenti-fideLinkages.Initially,wetestedtheaboveworkflowwithallfied.MetallatedionsformedbyabundanttrypticdisulfideMSandMS/MSdataobtainedbyawiderangeofcommonpeptidesthroughelectrosprayionization(ESI)werealsofounddigestionprotocolsunderalkalinepHconditions(seeTableandverifiedbytheirprecursormassandcoelution.TheS1)thatwereusedindevelopingNISTmAbspectralidentificationofthesedisulfidepeptidevariantsfacilitates5,6libraries.WethencompareddifferentdigestionprofilesreliabledisulfidebondanalysisandprovidesadditionalusingboththenumberandionintensitiesofconfidentfragmentationinformationasshownintheFragmentationidentificationsofallpeptideclassesincludingnativedisulfide-section.Table2summarizestheLC−MSanalysesofdisulfide-bondedpeptides(seeFigureS2A,B).Bothnon-reductionandlinkedpeptidesforeachlinkageandprovidesthenameofpartialreductionprotocolswereselectedasthepreferreddisulfidebonds,thelinkagelocationintheprotein,class,thedigestionconditionsunderwhichtheidentificationofrelativelynumberofmissed-cleavedsites,sequenceofpeptides,mass,higherabundancesandlargenumbersofdisulfide-containingobservedchargestates,andabsoluteionsignalintensities.peptideswereachieved.Fordevelopingspectrallibraries,theMajorDisulfideTrypticProducts.Disulfide-bondedpeptideexperimentsweredesignedtogeneratethewidestpossiblepairsareclassifiedasdisulfidetrypticpeptideswhenallterminivarietyofpeptidesofanalyticalconcern.Noattemptswereareconsistentwithsimpletrypticcleavage.Otherpeptidemadetooptimizedigestion;instead,weemployeddistinctbutvarietiessuchasoxidizedandmetallateddisulfidepeptidescancomplementaryprotocolstoachieveabroadcoverageoforiginatefrommodificationtothesepeptides.Table2showspeptidesandmodifications.Consideringthatdisulfidebondthatmajordisulfidetrypticpeptidesofseven(bonds2to6andshufflingcanbeeasilyinducedunderthesecommonalkaline8to9)outofninelinkagesproduced66to99%ofthetotalpHconditions,non-reducingandpartiallyreducingexperi-disulfidepeptideabundance(column3inboldofTable2).InmentsunderneutralpHforproteindigestionswereperformedcontrast,thepercentabundanceoftrypticpeptidesofbond1inordertocontrolshufflinginducedbyLC−MSanalysis,aswaslow(25%)andtrypticpeptidesofbond7wasnotdescribedintheMaterialsandMethodssection.detectable,indicatingtheneedforalternativepeptideclassesTheNISTmAb,atypicalIgG1protein,iscomposedoffour(e.g.,missedcleavageand/orirregularcleavage)foracompletepolypeptidechainsincludingtwoidenticalheavy(H)andtwoanalysisofdisulfidesinantibodies.identicallight(L)chains(FigureS1).EachHchainhasaMissedCleavageProducts.Disulfidebondsgenerally22variabledomain(VH)andthreeconstantdomains(CH1,CH2,hinderproteolyticdigestionbycommonenzymes;thus,andCH3),whileeachLchainhasavariabledomain(VL)andapeptideswithuptofourmissed-cleavagesitesweresought.constantdomain(CL).Intotal,theproteinhas12individualThisledtotheidentificationof152disulfide-bondedpeptidesstructuraldomains.Eachdomaincontainsasingledisulfidewithmissed-cleavagesites,whichaccountedfor40%ofthebondoranintrachaindisulfidebond.ThetwoHchainsaretotalidentifiedpeptides.AsseenfromTable2,thenumberoflinkedtoeachotherinthehingeregion(Hinge)bytwomissed-cleavagesitesareshowninColumn4andtheywereinterchaindisulfidebonds,andeachHchainisconnectedtoobservedinalldisulfidebondsexceptthebondCH3.VaryinganLchainbyaninterchaindisulfidebond(H−L).Thus,thelevelsoftrypticpeptideswithmissedcleavageareidentifiableNISTmAbhasatotalof12intrachaindisulfidebondsandfourwithmultiplechargestates.Thedetectionofsuchpeptidesisinterchaindisulfidebonds.Inthispeptidemappingstudy,thereanimportantsupplementarydisulfidedetermination,especiallyareninedistinctnativedisulfidebonds(Table2)thatneededwhentheirtrypticcounterpartsaretoosmalltobedetectedverificationfortheNISTmAbduetothesymmetrical(H−L,bond7)orlowabundancespecies(VH,bond1).Fourmolecularstructureformedbytheseantibodies.alternativedisulfidepeptidescontainingonetothreemissedDisulfideanalysesweredonebyidentifyingallpotentialcleavageswereobservedforthebondH−Latthemultipledisulfide-linkedpeptideclassesfromthetrypticdigestsofthechargestatesshowninTable2,ensuringconfidenceintheNISTmAb,includingfullytrypticcleavage,expectedmissedassignmentofthisdisulfidelinkage.Furthermore,thepresencecleavages,irregularcleavage,ESI-inducedadducts,andotherofreducedandalkylatedCysresidueswiththedisulfide-modifications.Mostdisulfide-containingpeptidesarecon-bondedpeptides(BondsH−LandHinge)canincreasetheirnectedbyasingledisulfidebond.Theexceptionisthepeptidesionsignals,andthustheirabundancespresentedinTable2areinthehingeregionthatcontaintwodisulfidebondslocatedinsignificantevenwithovernightdigestion.Missed-cleavagetheproximityofeachothertoconnecttwoHchains,asproductsproducedadditionalfragmentationpatternsandtheirdescribedintheMaterialsandMethodssection.Table2showsspectraarepartofthespectrallibrary.theninedistinctnativedisulfidebondsmappedintheIrregularCleavageProducts.Irregularcleavageproducts,NISTmAbthroughtheidentificationofthedisulfide-linkedinvolvingasinglesemitrypticcleavage,aresoughtduringthepeptidescorrespondingtoeachlinkage.ThetablepresentsMS1ionclustersearch.Intotal,27suchpeptides,arisingfromMS1peptidedataacquiredfromthetriplicateLC−MSmajortrypticpeptidecleavage,wereidentified,withmostofanalysesofthe18hsolutiontrypticdigestoftheNISTmAbthemgeneratedfromproteindigestion,whereasafewwerepartiallyreducedwithTCEPandalkylatedwithIAMatpH8.producedfromelectrosprayionization(ESI)in-sourceTheseidentifieddisulfide-linkedpeptidesrangeinmassfromfragmentation.Althoughtheseareminorproductsresulting1260to9888Dawithchargestatesfrom+2to+9.Alargefromlargerdisulfidespecies,theycanprovide,asshortmajorityoffullytrypticandmiscleaveddisulfidepeptidesarepeptides,usefulinformationtoaidinfragmentationanalysis.detectedwithmultiplecharges.Furthermore,morehighlyForexample,thesemitrypticpeptideions5to7inTable4arechargedionsoftrypticpeptideswithmissedcleavageareselectedforcharacterizingdisulfidebondfragmentationfound,providingcomplementaryinformationforthelinkagebecausetheirMS/MSspectraldatacontainusefulstructuralanalysis.DisulfidepeptidescontainingmultipleMet/Trpinformation.1618https://dx.doi.org/10.1021/acs.jproteome.0c00823J.ProteomeRes.2021,20,1612−1629

7JournalofProteomeResearchpubs.acs.org/jprArticleFigure2.SummedabundancesforeachoftheninenativedisulfidebondsintheNISTmAbobtainedbymedianresultsoftriplicateanalysesfromthreeseparatetrypticdigestsat0.25,2,and18husingpartiallyreducingandnon-reducingconditions.(A)PartiallyreducedpeptidemappingusinglowconcentrationofTCEPatpH8;(B)non-reducedpeptidemappingatpH8.ComplexMet/TrpOxidationMixturesofDisulfideSpecies.fromtwoLC−MSanalysesatpH8forpartiallyreducedandSeveralMet/Trp(methionine/tryptophan)oxidationproductsnon-reducedproteinsdigestedfor0.25,2,or18h.Thesite-areobservedwithtwodisulfidelinkagesofVHandCH3(bondsspecificquantitationcombinedtheionintensitiesofmultiply1and4,Table2)wheremultipleMet/Trparepresent,inthechargeddisulfidetrypticpeptidesandpeptideswithmis-partialreducingexperiments.Theyarelikelygeneratedfromcleavagesateachdisulfidebond.Itiswell-knownthatLC−MStwodifferentsources:in-sampleandin-source,whichisabundancemeasurementoflow-intensityions,suchas5,26consistentwithpreviousobservations.Oxidizedspeciesdisulfide-linkedions,isverychallenginggiventheintrinsicwithmorethanoneMet/Trpresidueoftenproduceamixturebiasagainstlowabundancepeptides,theirlowerionizationofdisulfide-linkedpeptidespectra.StructuraldifferencesoftheefficiencyinLC−ESI−MS,andpossiblerun-to-runvariationoxidationmixturesareduetopositionvariationsofMetorTrpinionsuppressionandothers.Weusedthesite-specificorbothinthesequence,makingthemdifficulttorecognizequantitationasameasureoftherelativedetectabilityoftheusingconventionalsoftwaretools.SelectedionchromatogramsninenativedisulfidebondsbytheLC−MSanalyseswiththeofoxidizeddisulfide-bondedpeptidescontainingmethionine/selectedtwoprotocols.Atshortdigestiontimes(0.25and2h),tryptophanareshowninFigureS3.TheirMS2mixturespectramostlylow-abundancespecieswereobservedandnodisulfide-aremanuallyvalidatedandcollectedinthelibrary.containingpeptidesweredetectedfromtheLchainvariableComparisonofTrypticDigestionProfilesofPartially/regionsaswellasfromthehingeregion.At18h,mostoftheNon-reducedNativeDisulfideBondsatpH8.Wemajordisulfide-linkedpeptideswereobservedwithsignifi-conductedacomparativeevaluationoftheabovetwoprotocolscantlyhighersignalintensitiesshowninFigure2Abutnotintoestimatetheirdigestionefficiencyfordisulfidemappingnon-reduceddatasets(Figure2B,excepttheCH2linkage),usingMS1ionsignalintensitiesoftheuniqueidentificationsofwhichmaysuggestthatalongertimeisneededtodigestnon-disulfide-containingpeptides.Thedigestionofpartiallyreducedproteins.reducedandalkylatedNISTmAbproducesthreegroupsofInthedigestiontimecourseexperimentsfor0.25,2,and18Cys-containingpeptides:majoralkylatedpeptides,relativelyhatalkalinepH,atotalof129nativedisulfide-bondedabundantdisulfide-linkedpeptides,andminorreducedpeptideswereidentifiedbythepartialreductionconditionwithpeptides,whereasthenon-reducedandnon-alkylateddigestthelowconcentrationofTCEP,whereas85ofsuchpeptidesalmostexclusivelyyieldspeptidescontainingdisulfidebonds.weredetectedusingnon-reducingandnon-alkylatingcon-Thiscomparisonwasconductedusingdisulfide-linkedditions.Thespectraoftheseidentificationsareincludedinthepeptides.Thesummedabundancesforeachlinkagesitespectrallibrary.AmajoradvantageofthepH8partialdisplayedinthebargraphfromFigure2A,Bwereobtainedreductionapproachisthatpartiallyreduceddisulfidesand1619https://dx.doi.org/10.1021/acs.jproteome.0c00823J.ProteomeRes.2021,20,1612−1629

8JournalofProteomeResearchpubs.acs.org/jprArticleFigure3.LevelsofscramblingoccurringatpH8foreachofCyssitesareobservedfromnon-reductionandpartialreductionexperiments.PartialRed.:reductionbyTCEPandalkylationbyIAM.Non-Red.:noreductionandalkylationforantibodydigestion.CysteinesarelabeledbytheirresiduenumberinLorHchain,e.g.,HC264denotesCysatresidue264oftheHchain.Figure4.Peptideidentificationscontainingnativedisulfidebondsobtainedfromfourtriplicateanalysesofthe18htrypticdigestusingdifferentparametersforfourprotocolvariations:non-reducingandpartiallyreducingconditionsatpH7andpH8,respectively.alkylatedcysteinesgreatlyimproveproteindigestionefficiencyeratedmuchlessscramblingascomparedtothepartial5forlinearandalkylatedpeptidesaswellasfordisulfide-reduction,althoughHC223andLC213showhigherlevelofcontainingpeptides.Thisalsoallowstheidentificationofscramblingat42and68%,respectively.Itisevidentthatwhendisulfide-bondedpeptidesalongwithanynon-disulfide-usingpH8forproteindigestion,whethernon-reducedorcontainingpeptidesinthesameexperiment.partiallyreducedsamples,widespreaddisulfiderearrangementDetectionofScrambled(Unexpected)Disulfidecouldoccur.LC213wasdetectedasahotspotforbothBondsatpH8intheLC−MS/MSAnalysis.Aftertheconditions.nativeNISTmAbdisulfidelinkageswereverifiedusingtheTominimizedisulfidebondrearrangementatbasicpH,twonon−/partiallyreducingLC−MS/MSanalyses,weappliedthesetsofcontrolexperiments(non-reductionandpartialsameworkflowtofinddisulfideswithunexpectedlinkages.Allreduction)wereperformedbyadjustingthebufferpHto7136possiblelinkagesthatcanbeformedfromthe16CysandusingNEMasthealkylatingreagentduetoitsthiolresiduesweresought.OnlyunmodifieddisulfidepeptidesarespecificityandstabilityatorbelowpH7,asdescribedinthesought.TheresultsrevealthatthesereactionsreadilyoccuratExperimentalSection.Over90%ofthedisulfidebondartifactsnearlyallCysresiduesunderalkalineconditions(pH8),withdetectedatpH8disappearedorwereonlyfoundattheMS1theexceptionofLC87.InadditiontothenineexpectednativetracelevelswhenneutralpHandNEMwereused.Sevendisulfidelinkages,86non-nativedisulfidelinkageswerepeptideswithscrambleddisulfidesidentifiedfromMS/MSobservedfrom109disulfide-scrambledtrypticpeptides.Figure3displaysthelevelofscramblingoccurringinthe18spectraareshowninTableS2A.Onlyfourlow-abundancehtrypticdigestatpH8foreveryCysresidueoftheNISTmAbnon-nativedisulfidebondsweredetectedinthenon-reducedasthepercentabundanceofthetotalintensitiesofalldisulfidedigest,andjustonewasdetectedinthepartialreductionbondsfromeachCysincludingnativeandscrambled.Inthedigest.SevenCysresidues(HC22,HC147,HC264,HC324,partialreductionoftheproteindigest,8outof16CysresiduesHC370,LC23,andLC213)areinvolvedintheformationofexhibitover15%disulfidescrambling.Inparticular,HC203,scrambleddisulfidebondsdetectedattracelevels.ThisresultLC193,andLC213comprise33,31,and72%ofalldisulfideillustratesthatsamplepreparationusingneutralorlowerpHabundances,respectively.Thenon-reductionconditiongen-significantlydecreasesshuffling.1620https://dx.doi.org/10.1021/acs.jproteome.0c00823J.ProteomeRes.2021,20,1612−1629

9JournalofProteomeResearchpubs.acs.org/jprArticleFigure5.DifferenttypesofproductionsintheMS/MSspectrumofatriplychargeddisulfide-linkedpeptide,SGTASVVCLLNNFYPR_SS_-YACEVTHQGLSSPVTK,atm/z1186.257withlessthan5ppmdeviationfromitstheoreticalvalue,fromFT-CIDfragmentationatNCEof35%.(A)Sequencingdisulfidelinkageand(B)fullyannotatedMS/MSspectrum.The*symbolingreencolorrepresentsfragmentsfromamidebondcleavagesandcontainstheintactdisulfidebond.TheannotationinredcolordenotesfragmentsfromC−Sbondcleavage.TogainabetterunderstandingofthepH7data,the(3)Applythistooltoallspectracontainingdisulfidebondsidentificationofnativedisulfide-containingpeptidesatpH7isthatwereidentifiedbyMS1ionanalysis.alsocomparedtothatatpH8.Figure4showsthenumberof(4)Developanevaluationtoolforassessingthethorough-disulfide-bondedpeptidesdetectedineachoffourtriplicatenessandaccuracyofannotationsbyexaminingthemassanalysesofthe18htrypticdigestundernon-reductionandaccuracyandabundanceratioofassigned/unassignedpartial-reductionatpH7andpH8,respectively.Thenotableproductions.variationinthenumberofidentifiedionsisobservedatalllinkagesbydifferentdigestionprotocols,althoughmore(5)Ifstep4findsincorrectormissedannotations,gobackagreeableresultsareseenatfourlinkages(CH1,CH2,CH3,tostep1toobtainnewknowledgeofunknownandCL).Inallcases,thepartialreductionatpH8yieldsmorefragments,andthenworkthroughsteps1to5untildisulfide-containingpeptidesthanothers.AtpH8conditions,nomoresignificantionsareunassigned.atotalof114differentionscontainingnativedisulfidebondsOverviewofDifferentTypesofFragmentsObserved.wereidentified,while55ofsuchionswereidentifiedunderpHInthissection,weintroducedifferenttypesofproductions7conditions(seeTableS2B).ItisevidentthatatpH7,eachcontainingadisulfidebondwithanexamplespectrum.Then,linkageyieldsfewerionsthanthoseatpH8;specifically,nowedescribethegeneralfragmentationfeaturesofHCDanddetectableionsarefoundattwooutoftheninedisulfideCIDspectraobtainedinFTMS/MSanalysisofdisulfide-linkages.TheseresultssuggestthatpHisacriticalparameterbondedpeptidesunderlowcollisionalenergy.foreffectivedisulfideanalysis,andtheselectionoftheFigure5showsanannotatedMS/MSspectrumofapeptidepreferabledigestionconditionsshouldconsiderthedetect-containinganintrachaindisulfidebond.Atotalof100uniqueabilityofbothnativeandscrambleddisulfidebondedpeptides.productionswereassigned,identifiedwithrelativeabundanceFragmentationAnalysisofDisulfide-LinkedPeptidesgreaterthan1%ofthebasepeak,whichconstituted92%ofthetotalionintensity.Table3summarizesthe55fragmentsAcentralobjectiveofthisworkwastocompletelymapallthehavingarelativeabundanceabove5%withamassaccuracydisulfidelinkagesintheNISTmAbbyidentifyingasmanywithin5ppm.Thesefragmentsareorganizedintothreegroupsdisulfide-bondedpeptidesandtheirproductionsaspossible.(seecolumn1ofTable3andTable1intheMaterialsandThiswasdoneinthefollowingmanner:Methodssectionforthedetailednotation).(1)ManuallyannotateMS/MSspectraofdisulfide-linkedInthisexample,group1contains35peptidebackbonepeptides.fragmentsallwithanintactdisulfidelinkage.Forexample,the(2)Developanidentificationtoolforautomaticfragmentdominantpeakatm/z1478.229ofgroup1a,asshowninassignmentusinginformationfromthepreviousstep.Figure5A,correspondstothedoublychargediony9ofP11621https://dx.doi.org/10.1021/acs.jproteome.0c00823J.ProteomeRes.2021,20,1612−1629

10JournalofProteomeResearchpubs.acs.org/jprArticleapercentabundanceVYACEVTHQGLSSPVTVYACEVTH10.2YACEVTHVYACEVTHQ19.5VYACEVTHQGLS32.847VYACEVTHQGLSVYACEVTHQGLSS30VYACEVTHQGLSS27.738.448.7VYACEVTHQGLSSPVVYACEVTHQGLSSPVVYACEVTHQGLSSPV18.2VYACEVTHQGLSSPVTVYACEVTHQGLSSPVT30.186.622.131.8VYACEVTHQGLSSPVTKVYACEVTHQGLSSPVTKVYACEVTHQGLSSPVTK6.9VYACEVTHQGLSSPVTK6.615.655.1VYACEVTHQGLVYACEVTHQGLSVVCLLNNFYPR744.111_SS__SS__SS__SS_VYACEVTHQGLSSPVTK_SS__SS__SS_17.7VYACEVTHQGLSSPVTK_SS_VYACEVTHQGLSSPVTK7.226_SS__SS__SS__SS__SS_VCLLNNFYPR8.1VYACEVTHQGLSSPVTKVYACEVTHQGLSSPVTK18.525.4VYACEVTHQGLSSPVTK18.7VYACEVTHQGLSSPVTKVYACEVTHQGLSSPVTK19.926.5VYACEVTHQGLSSPVTKVYACEVTHQGLSSPVTK98.5_SS__SS__SS_22.8_SS_VYACEVTHQGLSSPVTK_SS__SS__SS_13.1_SS__SS__SS_VYACEVTHQGLSSPVTK_SS__SS_VYACEVTHQGLSSPVTK5.56.4VYACEVTHQGLSSPVTK_SS_100_SS__SS__SS__SS__SS__SS__SS__SS__SS_33CLLNNFYPR3SVVCLLNNFYPRSVVCLLNNFYPR22SGTASVVCLLNNFYPRSGTASVVCLLNNFYPR2SGTASVVCLLN2SGTASVVCLLNN22SGTASVVCLLNNFYPRVCLLNNFYPR22SGTASVVCLLNNF2SGTASVVCLLNNFYPR2SGTASVVCLLNNF2VVCLLNNFYPRSVVCLLNNFYPR22SGTASVVCLLNNFY2SGTASVVCLLNNFY2SGTASVVCLLNNFYPR2SGTASVVCLLNNFY2SGTASVVCLLNNFYPRSGTASVVCLLNNFYPR2SGTASVVCLLNNFYPR11YPRPVTKO3SGTASVVCLLNNFYPRO2O2SGTASVVCLLNNFYPR2SGTASVVCLLNNFYPR3O2]10]SGTASVVCLLNNFYPR]2]112SVYACEVTHQGLSS2SGTASVVCLLNNFY2SGTASVVCLLNNFY2VYACEVTHQGLSSPVYACEVTHQGLSSPVTK2O2SGTASVVCLLNNFYPR22111223-H2NO2-HSGTASVVCLLN-2H33NO32-NHSGTASVVCLLNNFY-H-y:y+S+S2VYACEVTHQGLSSPVTK1VYACEVTHQGLSSPVTKO-H25SGTASVVCL51314-Hde-LinkedPeptide,SGTASVVCLLNNFYPR_SS_VYACEVTHQGLSSPVTK162889121213131515151616:b(2):b(2)1717+S+S17411SVVCLLNNFYPRSGTASVVCLLNNFYPRfifragment-NHcharge-H-Hsequence-NH-NH-H-NH141491212911111210131311121414141412163assignmentofppmmasserrorinz/m985.82231.1y925.93943.3435.2348444.2815y(2)0.60.4y1075.2041080.8791131.54911308.64211.91312.12651.42.21321.130911385.15951y1413.68710.3y1431.2117b(2)0.21478.2294.61488.2318b0.41504.7283b(2)b(2)1513.73250.5b(2)1.31527.7645b3.31539.2399b2.61553.25881.4y91557.2498b3.81561.7668b(2)2.71577.3018b(2)3.31612.3059y0.71625.7781b(2)3.31629.30112b4.11634.7865b(2)1.81641.3032CLLNNFYPRb0.61643.2998y0.11646.7877y2.81655.3022b0.11696.8263a2.61705.8307b1.8a(2)1.51306.1199b11329.6398b(2)1373.158b(2)4.11404.179b(2)1.2b(2)2.81456.71364.9[b(2)1772.8417[a1850.88614.1[a2.3[b(2)4.6yyy(2)peptidepeptidespeptides1b1cdoubleC-NcleavageinonepeptidedoubleC-Ncleavageinboth1533.75212a3.1C-Scleavageint5-153asingleC-Ncleavageinboth2892.9614SVVCLLNNFYP1.2classgroupcleavagesitefragmentsbondTable3.FragmentIonAnnotationinanFT-CIDMS/MSSpectrumoftheCharge3+Disulintactdisulfidebondfragment1asingleC-Ncleavageineithercleaveddisulfidebondfragmentswithoutdisulfide1622https://dx.doi.org/10.1021/acs.jproteome.0c00823J.ProteomeRes.2021,20,1612−1629

11JournalofProteomeResearchpubs.acs.org/jprArticlelinkedtoP2viaadisulfidebond.Asidefromtheseionsgeneratedbysinglepeptidebondcleavage,morecomplexproductsarisefromdoublecleavagesuchastheionsinthepercentabundancegroups1band1c.Forinstance,thepeakatm/z1533.752formedbydoublecleavageofamidebondswasidentifiedasthedoublychargedioninvolvingtheP1residuesfrom5to15linkedtoP2viaadisulfidebond,labeledInt5_15inGroup1bofFigure5A.Anotherexampleisthedoublychargedinternalionatm/z1373.158consistingofthefragmenta14ofP1andthefragmentb(2)12ofP2connectedbyadisulfidebond(Group1cofFigure5A).Overall,group1comprisesamajority(71%)oftotalproductionintensityandprovidesdetailedpeptidesequence.ThisexamplespectrumalsocontainsfiveproductionsarisingfromdisulfidecleavageasshowninredcolorinFigure5BandGroup2aofTable3.TheywereformedthroughthelossofH2S(e.g.,charge2+y(2)17-H2S)ortheadditionofS(e.g.,thesinglyanddoublychargediony(2)17+S)intheC−Sbondcleavageproducts.Group3includes15standardpeptidefragmentswithoutadisulfidebond.Someofthemarehighlyabundant,suchasthesinglychargedy6ofP1andy(2)11ofP2.Thisgrouprepresentscommonbackbonefragments,usefulforsequencingbothP1andP2.11SPVTK1FYPR1SSPVTK1NFYPR1GLSSPVTK1NNFYPR1QGLSSPVTK1QGLSSPVTK1LNNFYPR1LLNNFYPR1HQGLSSPVTK1THQGLSSPVTKEVTHQGLSSPVTK7.3108.821.218.513.551.81240.929.92344.511.5KeyFeaturesofDisulfideLinkageFragmentationcproductionswithrespecttothebasepeakinaMS2spectrum.fiPatterns.Low-energycollisiondissociationofdisulfide-containingpeptidesgeneratesproductions,whichcontain3intact,cleaved,andnodisulfidebonds.CleavageofanS−S-NHbondleadstotheformationoffragmentscontainingfreeCys56899101113fragment4charge567sequence8orCysthioaldehyde(−2Da)whilecleavageoftheC−SbondassignmentofyieldsfragmentswithCyspersulfide(+32Da)ordehydroa-themass/chargeratioforfragmention.Errorppm:massdeviationbetweenobservedandtheoreticallanine(−34Da).Thedissociationofamidebondscanproducetypicalb/yions,withorwithoutintactandcleavedm/z:disulfidebonds.ppmWeconductedacomprehensiveanalysisofdisulfidebondsunderfragmentationbylowenergycollisiondissociationfor15masserrorindebond.fimultiplychargeddisulfide-linkedpeptideions(Table4).Theyincludeionsoftryptic,semitryptic,andmiscleavedpeptides.z/DifferenttypesoffragmentionsarisingfromthedisulfidebondmwereexaminedusingspectraacquiredatNCEvaluesof16%,531.3135582.3021618.34590.4696.34652.3788.45030.3810.38890.2899.4845y(2)1.2916.511y0.5923.4728y(2)1.4y1.3y(2)0.6yy(2)y(2)y1036.55771053.56771154.61550.31382.723110.83.1yy(2)y(2)y(2)20%,and24%forHCDaswellas35%forFT-CID.Thelastcolumnshowsthenumberofreplicatespectrausedinthecalculationofmedianvalues.Threetypesoffragmentationpatternswereobserved,(1)amidebondcleavageproductscontaininganintactdisulfidebondarepredominantfragments;(2)disulfidebondcleavagesleadingtominoryetextensivefragmentation;and(3)doublecleavageproductsarecommonandsignificantfragments.Thesekeyfeaturesaremorefullydescribedbelow.KeyFeature1:FragmentsContaininganIntactDisulfideBondPredominateunderHCDandFT-CID.Forthepurposeoffindingmajorfragmentsindisulfidespectra,weexaminedavarietyofproductionsanddividedthemintothreecategories,asshowninFigure6.Oneinvolvesfragmentscontaininganintactdisulfidebond,labeledDSBfragmentsinthisanalysis,thesecondarefragmentsnotcontainingCys,labelednon-DSBfragments,andthelastsetincludesallunassignedpeaks.DSBfragmentsmakethelargestrelativecontributionintheclassgroupcleavagesitespectraforallpeptidesstudied,witheachyieldingfrom46to76%oftotalproductionintensity.Non-DSBfragmentsfrompeptidebondcleavageaccountedfor14to44%oftotalionvalues.z:chargestateoffragmention.Sequence:sequenceoffragmention.Abund.%relativeabundanceofspeciintensityinthespectra,providingcomplementarysequencingTable3.continuedaNotes:Classandgroup:fragmentclassandgroup.Cleav.site:peptidebondordisulm/zinformationtoDSBfragments.Theunassignedpeaksappeared1623https://dx.doi.org/10.1021/acs.jproteome.0c00823J.ProteomeRes.2021,20,1612−1629

12JournalofProteomeResearchpubs.acs.org/jprArticleTable4.SelectedDisulfide-LinkedPeptideIonswithVariedAminoAcidCompositionsandChargeStatesthatWereStudiedforFragmentationPatternsunderHCDandFT-CIDbondCyspositionpeptide#precursorreplicate#bondnameinproteindisulfide-linkedpeptidesequenceclassionchargem/zspectra2CH1147−203STSGGTAALGCLVK_SS_DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYStryptic161320.49442LSSVVTVPSSSLGTQTYICNVNHKPSNTK271131.9961573CH2264−324TPEVTCVVVDVSHEDPEVK_SS_CKtryptic33777.0442444583.032955*EVTCVVVDVSHEDPEVK_SS_CKsemi-53711.00618*TPEVTCVVVDVSHED_SS_CKtryptic63625.8542074469.717144CH3370−428NQVSLTCLVK_SS_WQQGNVFSCSVMHEALHNHYTQKtryptic84962.21314995769.972582106641.81141117550.268296CL133−193SGTASVVCLLNNFYPR_SS_VYACEVTHQGLSSPVTKtryptic1231186.25717134889.945277145712.1574757H-L223−213SCDK_SS_SFNRGECmissed153421.16930cleavageaNotes:Thesymbol*indicatessemitrypticpeptides.#bondandbondname:sequentialnumberandnameofbondsgivenbythiswork.Cyspositioninprotein:Cysresidueinproteinsequence.Peptideclass:trypticdigestproducts.#ion:sequentialnumberofselectedions.Charge:chargestates.Precursorm/z:themass/chargeforpeptideionsinMS1.Replicatespectra:repeatspectraidentifications.Figure6.Comparisonofthreeioncategoriesfrom15disulfide-linkedpeptideions(Table4)bypercentionintensity.DSBfragments:ionslinkedbyadisulfidebond.Non-DSBfragments:ionsnotcontainingadisulfidebond.Unassigned:unassignedions.Figure7.Distributionofdisulfide-bondcleavagesamongfivedisulfidelinkagesobtainedfrom3200high-qualityMS2spectra.Pksreferstofragmentionsarisingfromdisulfidecleavage.toprimarilyarisefromlow-abundancefragmentsthatcannotbonds)complementsdisulfideconnectivityinformation.Suchberecognizedduetothelackofisotopicdata.productionshavebeenreportedasrarecomparedtoamideKeyFeature2:ExtensiveDisulfide-BondCleavagebondbreakageviathegasphasefragmentationofdisulfide-27,28ObservedatLowLevels.Disulfide-bondfragmentationlinkedpeptidesbylow-energycollisiondissociation.IninvolvingCyssidechainbreakage(i.e.,C−SandS−Scontrast,weobservedextensivecleavageproducts,althoughat1624https://dx.doi.org/10.1021/acs.jproteome.0c00823J.ProteomeRes.2021,20,1612−1629

13JournalofProteomeResearchpubs.acs.org/jprArticleFigure8.MS2analysisofthecharge/energy-dependencechangesinHCDfragmentationofS−SandC−Sbondsofdisulfide-linkedpeptide,TPEVTCVVVDVSHEDPEVK_SS_CKat(A)m/z583.032ofthecharge4+ionatanNCEof20and(B)m/z466.627ofthecharge5+ionatanNCEof24.Theintensityaxisisscaleddowntoanintensityrangefrom0to12%relativetothebasepeaktoshowlowlevelsofdisulfidebondcleavageintheHCDspectrum.*fragmentscontainanintactdisulfidebond.C−SandS−Sindicatecleavagefromdisulfidebonds.Figure9.HCDspectrumofthecharge7+disulfide-linkedpeptideion,NQVSLTCLVK_SS_WQQGNVFSCSVMHEALHNHYTQK,atNCE=24showsdominantdoublecleavagethatgeneratedanumberofmajorinternalfragmentscontainingadisulfidebond(insquarebrackets[]).*fragmentscontainanintactdisulfidebond.lowlevels,arisingfrombothS−SandC−Sbonds,includingyieldprevalentdisulfidebondcleavage.Over93%(H−L)andfreeCys,Cysthioaldehyde(−2Da),Cyspersulfide(+32Da),60%(CH2)oftheirtotalspectracontain4to15cleavageanddehydroalanine(−34Da).productsarisingfromC−SbondsandS−Sbonds.ThisisFigure7showsthedistributionofobserveddisulfidebecausethetwositesaremadeupofsmallerpeptides:cleavageproductsresultingfromover3200good-qualitySCDK_SS_SFNRGECandTPEVTCVVVDVSHED-MS2spectraofthefivedisulfidebondsstudiedinTable4.PEVK_SS_CKanditssemitrypticpeptides,whicharemoreThespectraweredividedintofourgroupsintermsofthepronetoundergoingS−SandC−Sbondcleavages.Inournumberofdisulfidecleavageproductsobserved(e.g.,peaks=0data,fragmentsresultingfromdisulfidecleavageweretypicallyfornopeaksfromdisulfidecleavage;≤3;between4to7;andobservedinlowabundance;forexample,thesummed>7).ThepercentagesofspectraineachgroupovertotalabundanceoftheC−SandS−Sbondcleavageisfrom1toreplicatespectra(Column9ofTable4)werecalculatedfor5%ofthetotalproductionintensity.eachbondlinkage.ThesedataindicatethatmostofthelinkageWeobservedmoreionsarisingfromS−Sbondcleavageatsitesproduceduptothreefragmentsresultingfromdisulfidelowerchargestates,whereasmoreionsfromC−Sbondfragmentation,whiletwodisulfidelinkages(CH2andH−L)cleavagewereobservedathigherchargestates.Figure81625https://dx.doi.org/10.1021/acs.jproteome.0c00823J.ProteomeRes.2021,20,1612−1629

14JournalofProteomeResearchpubs.acs.org/jprArticleFigure10.Head-to-tailplotofexperimentalandreferenceHCDtandemmassspectraofanioncontaininganativedisulfidebondCH2.ItshowsanexperimentalESI-MS/MSspectrumacquiredfrom2DLC−MS/MSanalysisofreducedhumanplasmareferencematerialNISTSRM1950matchingtothecharge4+disulfide-linkedpeptidespectruminthelibrary,“TPEVTCVVVDVSHEDPEVK_SS_CK”.Thelibraryspectralsimilarityscoreis930(1000asthehighest).demonstratesthecharge/energy-dependentchangesintheconsensusmassspectra.Thespectrallibrarycontains702HCDfragmentationofS−SandC−Sbondsinthecharges4+consensusspectrafor149nativeand234scrambleddisulfide-and5+TPEVTCVVVDVSHEDPEVK_SS_CK.Theirfullybondedpeptideions(e.g.,sequence/modification/charge).annotatedMS/MSpeaklistscanbefoundinTableS3A,B.Eachentryinthelibraryprovidesinformationonprotein,CysMoreintenseS−Sbondcleavageisevidentincharge4+atpositioninsequence,disulfidebonds,precursorm/z,HCDNCE=20inFigure8A,whilemoreC−Sbondcleavagenormalizedcollisionenergy,identificationscore,percentagesproductsareseeninthecharge5+atHCDNCE=24inofassigned/unassignedabundances,andnumberofreplicateFigure8B.Thisobservationsuggeststhat(a)thenumberofmobileprotonsthatarerequiredtoinducedissociation29spectrausedforcreatingtheconsensusspectrum.PeaksinappearstohaveasignificanteffectonC−Sbondcleavageandeachspectrumarethoroughlyannotated.Thislibraryisfreely(b)withfewermobileprotonsandlowerNCEvalues,theS−Savailableathttps://chemdata.nist.gov/dokuwiki/doku.php?id=bondmaytendtoyieldmorecleavageascomparedtotheC−Speptidew:lib:disulfidepeptides.bond.UseoftheLibraryofDisulfide-LinkedSpectrainKeyFeature3:DoubleCleavageofDisulfide-LinkedGeneralAntibodyAnalysis.OnepotentialutilityofthePeptideIonsIsCommon.Theyarefrequentlyobservedforallmassspectrallibraryistoevaluatetheeffectivenessofdisulfidepeptidesinvestigatedinthisstudy.Thepercentreductioninpeptidemappingsamplepreparation.Weabundancesofdoublecleavageionsrangefrom10to35%ofdemonstratethisusingLC−MS/MSresultsfromthreetotalproductionintensity.Theformationofdisulfide-bondeddifferentdatasets,allofwhichwereperformedunderdoublecleavageionscanbequitecomplex.Theycanbeconventionalreducingconditions:Humira(acommerciallygroupedintotwocategories.Oneinvolveseitherdouble30cleavageofonepeptide(internalions)oracleavagefromeachavailablemAbdrug),humanplasma(aNISTreference31,32oftwocomponentpeptides,andtheotherresultsfromthematerial,SRM1950),andNISTmAbpreparedwithout5cleavageofbothpeptidebackboneanddisulfidebonds(seesecondreduction.NotethattheformertwosampleswereGroup2ofTable1).Followingbackbonefragmentation,preparedwithasecondreductionstep.Thisanalysiswasfurtherfragmentationoftheresultantfragmentsmaybe33carriedoutusingtheNISTMSQCPipeline.Itidentified5,derivedbythebreakageofthedisulfidebond.Thenumberand11,and29disulfide-linkedpeptidespectrainHumira,NISTabundancelevelofdoublecleavageproductsareenergy-andSRM1950,andNISTmAb,respectively,usingamatchscorecharge-dependent.Asexpected,highercollisionenergyandthresholdof450andmasserroroflessthan10ppm.Allchargevaluesappeartoincreasetheportionofdoubleidentificationsweremanuallyconfirmedandareshownincleavages.AsillustratedinFigure9,dominantdoublecleavageTableS4.Figure10showsanexampleofacharge4+peptideproductswereobservedintheHCDspectrumofthecharge7+CH3disulfidepeptide.Thelargeproportionofmultiplyioncontaininganativedisulfidelinkage,CH2detectedinthechargedmajorproducts(showningreencolor)observedin2DLC−MS/MSanalysisofreducedhumanplasmareferencethehighm/zregioncanbeassignedasarisingfromthematerial.InthereducedHumiraexperiments,wefoundtwosequentialcleavageofbothcomponentpeptides.Theproductnativedisulfidebonds.IntheNIST1950plasmareferenceionswereanalyzedforthepeptidein4+,5+,6+,and7+chargematerial,twonativeandtwoscrambledlinkageswerefound.states,showingthattherelativeabundancesofdoubleFurthermore,intheNISTmAbanalysis,whichdidnotincludecleavagesateachchargeare1,4,10,and22%,respectively.asecondreductionstep,allfivenativedisulfidebondsofConsensusMassSpectralLibrary.Wecompiledalibraryconstantregionsaswellasfourscrambleddisulfidebondswereofconsensusmassspectraofalldisulfide-linkedpeptideionsdetected.Theseobservationssuggestthat(1)lowlevelsofacquiredinthisstudyfromthetrypticdigestoftheNISTmAb.disulfide-linkedionscanbeidentifiedevenafterreductioninEachofthemarisesfrommultiplespectraofthesamepeptideLC−MSanalysisand(2)thesecondreductionstepoftenionatagivencollisionenergy.Theywerederivedfromthe34LC−MS/MSanalysisof82runsundernon-reducingandimplementedinstandardpeptidemappingexperimentsispartialreducingconditions.Spectrawereincludedonlyifatjustifiedasfewerdisulfidebondsweredetectedwhenthisextraleast60%oftotalpeakabundancecouldbeconfidentlyreductionstepwasdoneintheHumiraandNISTSRM1950annotated.Over10,000replicatespectraunderlietheseanalyses.1626https://dx.doi.org/10.1021/acs.jproteome.0c00823J.ProteomeRes.2021,20,1612−1629

15JournalofProteomeResearchpubs.acs.org/jprArticle■CONCLUSIONSbondedpeptidesfromthreeLC−MS/MSanalysesInthisstudy,weperformedadetailedanalysisofdisulfide-underreducedconditions(TableS4)(xlsx)linkedpeptidesgeneratedinthetrypticdigestionofareferencemonoclonalantibody,theNISTmAb.Thiswasdonebothfor■AUTHORINFORMATIONunderstandingthefragmentationofthesecomplexdisulfide-CorrespondingAuthorlinkedpeptidesandforcreatingareferencemassspectrallibraryforhelpingotherstoreadilyidentifythesepeptides.QianDong−BiomolecularMeasurementDivision,NationalForthispurpose,adataanalysisworkflowofcombiningMSInstituteofStandardsandTechnology,Gaithersburg,andMS/MSwasdeveloped,usingMSdatatoassignallMaryland20899,UnitedStates;orcid.org/0000-0001-possibledisulfide-containingpeptidespectraandthenMS/MS6466-6045;Phone:301-975-2569;Email:qian.dong@datatoresolveanyambiguousidentifications.Thisapproachnist.govhelpscircumventthetediousmanualinspectionofMS/MSAuthorsspectranecessaryfortheproteindisulfidebondprofilewithhighsensitivityandaccuracy.BecausethemethodsearchestheXinjianYan−BiomolecularMeasurementDivision,NationalcompletecombinationofallpossibledisulfidebondsinInstituteofStandardsandTechnology,Gaithersburg,proteinsbasedontheirsequence,allnativeaswellasnon-Maryland20899,UnitedStates;orcid.org/0000-0003-nativedisulfide-containingpeptidesarecoveredinsearches.3204-7420Hence,thisapproachcouldbeusefulforroutinedisulfideYuxueLiang−BiomolecularMeasurementDivision,Nationalmappingingeneralproteomicsapplications.Inourstudy,thisInstituteofStandardsandTechnology,Gaithersburg,ledtotheidentificationandannotationof428disulfide-linkedMaryland20899,UnitedStatespeptideionswithavarietyoforigins:fullytryptic,missedSanfordP.Markey−BiomolecularMeasurementDivision,cleavages,irregularcleavages,containingMet/Trpoxidation,NationalInstituteofStandardsandTechnology,andmetallatedadducts.Basedontheseresults,weeffectivelyGaithersburg,Maryland20899,UnitedStatescharacterizedtherelativelevelofalltheninenativedisulfideSergeyL.Sheetlin−BiomolecularMeasurementDivision,bondsanddetected86additionaldisulfidelinkagesfromNationalInstituteofStandardsandTechnology,shufflingintheLC−MS/MSanalysisoftheNISTmAb.AsGaithersburg,Maryland20899,UnitedStatesexpected,theS−SbondshufflingatpH8wasgreatlyConcepcionA.Remoroza−BiomolecularMeasurementminimizedunderneutralconditionpH7.Division,NationalInstituteofStandardsandTechnology,ThisanalysishighlightstheuniquedifficultiesinaGaithersburg,Maryland20899,UnitedStates;orcid.org/comprehensiveanalysisofdisulfide-linkedpeptidesespecially0000-0003-1540-1635wherereductionisincomplete,eitherbydesignornot,andWilliamE.Wallace−BiomolecularMeasurementDivision,wherefragmentationlacksreadilyidentifiablediagnosticNationalInstituteofStandardsandTechnology,fragmentions.TransformingtheseresultsintoaspectralGaithersburg,Maryland20899,UnitedStateslibrarywillenableotherstoefficientlyidentifybothnativeandStephenE.Stein−BiomolecularMeasurementDivision,scrambleddisulfidepeptidesinroutineproteomicsanalysesNationalInstituteofStandardsandTechnology,containingIgG1antibodies.WeshowedthatwithareferenceGaithersburg,Maryland20899,UnitedStateslibraryofconfidentlyassignedspectra,theunambiguousCompletecontactinformationisavailableat:assignmentoftandemmassspectra(MS/MS)todisulfide-https://pubs.acs.org/10.1021/acs.jproteome.0c00823linkedpeptidesequencescanbeeasilydone.WealsodemonstratedthatthislibrarycanidentifydisulfidebondsofNotesIgG1proteinsinreducedhumanserumdigests,therebyTheauthorsdeclarenocompetingfinancialinterest.providingauniquemeasureoftheeffectivenessofreductionmethodsinproteomicsexperiments.■ACKNOWLEDGMENTS■ThisworkwassupportedsolelywiththeNISTfunds.CertainASSOCIATEDCONTENTcommercialinstrumentsormaterialsareidentifiedinthis*sıSupportingInformationpapertospecifytheexperimentalprocedureadequately.SuchTheSupportingInformationisavailablefreeofchargeatanidentificationisnotintendedtoimplyrecommendationorhttps://pubs.acs.org/doi/10.1021/acs.jproteome.0c00823.endorsementbytheNationalInstituteofStandardsandSchematicdiagramoftheNISTmAbstructureasaTechnology,norisitintendedtoimplythatthematerialsorhumanizedIgG1κprotein(FigureS1);comparisonof15instrumentsidentifiedarenecessarilythebestavailableforthetrypticdigestionprotocolsusingpeptideclassanalysispurpose.Themassspectrometryproteomicsdatahavebeen35(FigureS2);selectedionchromatogramsofoxidizeddepositedtotheProteomeXchangeConsortiumviathe36disulfide-bondedpeptides(FigureS3);parametersusedPRIDEpartnerrepositorywiththedatasetidentifierin15protocolvariationsduringdenaturing,reduction,PXD023358.andalkylationof1mgoftheNISTmAb(TableS1);listofscrambledandnativedisulfide-bondedpeptides■ABBREVIATIONSPAGEidentifiedfromcontrolledexperimentsofthe18hC−S,carbon−sulfurbond;Cys,cysteine;DTT,dithiothreitol;digestionunderpartial/non-reductionatpH7(TableFT,Fouriertransformmassspectrometry;HCI,hydrochloricS2);annotatedpeaklistfortheHCDtandemmassacid;HCD,higher-energycollisiondissociation;IAM,spectrumofthecharge4+and5+disulfide-linkediodoacetamide;IgG1,subclass1ofimmunoglobulinGpeptide,TPEVTCVVVDVSHEDPEVK_SS_CK(Table(IgG1,2,3,and4);LC−MS,liquidchromatography−massS3);librarysearchofnativeandscrambleddisulfide-spectrometry;LC−MS/MS,liquidchromatography−tandem1627https://dx.doi.org/10.1021/acs.jproteome.0c00823J.ProteomeRes.2021,20,1612−1629

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17JournalofProteomeResearchpubs.acs.org/jprArticleMcCoy,L.F.;Momin,S.S.;Paladugula,N.;Pendergrast,E.C.;Pfeiffer,C.M.;Powers,C.D.;Rabinowitz,D.;Rybak,M.E.;Schleicher,R.L.;Toombs,B.M.;Xu,M.;Zhang,M.;Castle,A.L.DevelopmentofaStandardReferenceMaterialformetabolomicsresearch.Anal.Chem.2013,85,11732−11738.(32)Simón-Manso,Y.;Lowenthal,M.S.;Kilpatrick,L.E.;Sampson,M.L.;Telu,K.H.;Rudnick,P.A.;Mallard,W.G.;Bearden,D.W.;Schock,T.B.;Tchekhovskoi,D.V.;Blonder,N.;Yan,X.;Liang,Y.;Zheng,Y.;Wallace,W.E.;Neta,P.;Phinney,K.W.;Remaley,A.T.;Stein,S.E.MetaboliteprofilingofaNISTStandardReferenceMaterialforhumanplasma(SRM1950):GC-MS,LC-MS,NMR,andclinicallaboratoryanalyses,libraries,andweb-basedresources.Anal.Chem.2013,85,11725−11731.(33)NationalInstituteofStandardsandTechnology.TheNISTlibrariesofpeptidetandemmassspectraandavailablewindowssoftwareforpeptidemassspectrallibraries.Washington(DC):UnitedStatesDepartmentofCommerce;2016,http://chemdata.nist.gov/dokuwiki/doku.php?idDpeptidew:cdownload,andhttp://chemdata.nist.gov/dokuwiki/doku.php?idDpeptidew:msqcpipeline.Aug8.(34)Rebecchi,K.R.;Go,E.P.;Xu,L.;Woodin,C.L.;Mure,M.;Desaire,H.Ageneralproteasedigestionprocedureforoptimalproteinsequencecoverageandpost-translationalmodificationsanalysisofrecombinantglycoproteins:applicationtothecharacter-izationofhumanlysyloxidase-like2glycosylation.Anal.Chem.2011,83,8484−8491.(35)Deutsch,E.W.;Bandeira,N.;Sharma,V.;Perez-Riverol,Y.;Carver,J.J.;Kundu,D.J.;García-Seisdedos,D.;Jarnuczak,A.F.;Hewapathirana,S.;Pullman,B.S.;Wertz,J.;Sun,Z.;Kawano,S.;Okuda,S.;Watanabe,Y.;Hermjakob,H.;MacLean,B.;MacCoss,M.J.;Zhu,Y.;Ishihama,Y.;Vizcaíno,J.A.TheProteomeXchangeconsortiumin2020:enabling‘bigdata’approachesinproteomics.NucleicAcidsRes.2020,48,D1145−D1152.(36)Perez-Riverol,Y.;Csordas,A.;Bai,J.;Bernal-Llinares,M.;Hewapathirana,S.;Kundu,D.J.;Inuganti,A.;Griss,J.;Mayer,G.;Eisenacher,M.;Pérez,E.;Uszkoreit,J.;Pfeuffer,J.;Sachsenberg,T.;Yılmaz,Ş.;Tiwary,S.;Cox,J.;Audain,E.;Walzer,M.;Jarnuczak,A.F.;Ternent,T.;Brazma,A.;Vizcaíno,J.A.ThePRIDEdatabaseandrelatedtoolsandresourcesin2019:improvingsupportforquantificationdata.NucleicAcidsRes.2019,47,D442−D450.1629https://dx.doi.org/10.1021/acs.jproteome.0c00823J.ProteomeRes.2021,20,1612−1629