Identi fi cation of Deamidated Peptides in Cytokine-Exposed MIN6 Cells through LC − MSMS Using a Shortened Digestion Time and Inspection

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pubs.acs.org/jprTechnicalNoteIdentificationofDeamidatedPeptidesinCytokine-ExposedMIN6CellsthroughLC−MS/MSUsingaShortenedDigestionTimeandInspectionofMS2SpectraAïshaCallebaut,RitaDerua,SaurabhVig,ThomasDelong,ChantalMathieu,andLutOverbergh*CiteThis:J.ProteomeRes.2021,20,1405−1414ReadOnlineACCESSMetrics&MoreArticleRecommendations*sıSupportingInformationABSTRACT:Enzymaticdeamidation,theconversionofglutamine(Gln)intoglutamicacid(Glu)residues,mediatedbytissuetrans-glutaminaseenzymes,canprovokeautoimmunitybygeneratingalteredself-epitopes,aprocesswell-knowninceliacdiseaseandmorerecentlyalsodescribedintype1diabetes(T1D).Toidentifydeamidatedproteins,liquidchromatography−tandemmassspectrometryisthemethodofchoice.However,asnonenzymaticdeamidationsonasparagine(Asn)andtoaminorextentonGlnarefrequentlyinducedinvitroduringproteomicssamplepreparation,theaccuratedetectionofinvivodeamidationcanbehampered.Herewereportontheoptimizationofamethodtoreduceinvitrogenerateddeamidationby70%usingimprovedtrypsindigestionconditions(90min/pH8).WealsopointtothecriticalimportanceofmanualinspectionofMS2spectra,consideringthatonly55%ofthehighqualitypeptideswithGlndeamidationwereassignedcorrectlyusinganautomatedsearchalgorithm.Asproofofprincipal,usingthesecriteria,weshowedasignificantincreaseinlevelsofbothAsnandGlndeamidationincytokine-exposedmurineMIN6β-cells,paralleledbyanincreaseintissuetransglutaminaseactivity.Thesefindingsaddevidencetothehypothesisthatdeamidationisoccurringinstressedβ-cellproteinsandcanbeinvolvedintheautoimmuneprocessinT1D.KEYWORDS:enzymaticdeamidation,artifactualdeamidation,type1diabetes,autoimmunity■INTRODUCTIONanautoimmunediseasecharacterizedbyimmune-mediatedEnzymaticdeamidation,theconversionofglutamine(Gln)destructionofinsulin-producingβ-cellsintheisletsofintoglutamicacid(Glu)residues,mediatedbytrans-Langerhansinthepancreas,deamidationisthoughtto2,3glutaminase(TGM)enzymes,isacalcium-dependentprocesscontributetotheautoimmuneprocess.thatresultsinanincreaseinmassof0.984Da(Scheme1).Assuch,VanLummeletal.describedimmuneresponsestoAlthoughdeamidationisobservedinnormalphysiologicaladeamidatedproinsulinepitopeinT1DpatientsandhealthyDownloadedviaKINGABDULLAHUNIVSCITECHLGYonMay14,2021at16:05:34(UTC).processes,abnormaldeamidation,oftenassociatedwithcontrols,withmostoftherespondingpatientsproducingIFN-Seehttps://pubs.acs.org/sharingguidelinesforoptionsonhowtolegitimatelysharepublishedarticles.inflammation,canprovokeT-andB-cellautoreactivitybyγ,whereasresponsesinhealthydonorsweredominatedbyIL-4generatingalteredself-epitopes,awell-knownprocessinceliac10.McGintyetal.demonstratedthepresenceofmemoryT-disease(CD),wheredeamidatedglutenproteinsarethoughtcellsspecifictodeamidated65kDaglutamicaciddecarbox-1tobecausativeforthedisease.Alsointype1diabetes(T1D),ylasepeptidesatsignificantlyhigherfrequenciesexvivointheperipheralbloodofT1DpatientscomparedwithhealthyScheme1.OverviewofEnzymaticDeamidationa5controls.Inadditiontotheirdeamidationfunction,TGMsalsocatalyzethecross-linkingofproteinsbytheformationof6isopeptidebondsbetweenGlnandlysineresidues.Thiscross-linkingactivityhasalsobeenlinkedtoT1Dpathogenesis.Received:October10,2020Published:December29,2020aEnzymaticdeamidation,mediatedbytransglutaminase(TGM)enzymesleadingtotheconversionofglutamineintoglutamicacid.©2020AmericanChemicalSocietyhttps://dx.doi.org/10.1021/acs.jproteome.0c008011405J.ProteomeRes.2021,20,1405−1414

1JournalofProteomeResearchpubs.acs.org/jprTechnicalNoteaScheme2.OverviewofEnzymaticandNonenzymaticDeamidationa(A)Chemicalreactionoftheenzymaticallymediatedconversionofglutamineintoglutamicacidbytransglutaminaseenzymesthroughanacyl-enzymeintermediate.(B,C)Chemicalreactionofnon-enzymaticallymediatedconversionofasparagine(B)throughasuccinimideandglutamine(C)throughaglutarimideintermediateintoisoasparticacidandasparticacidandisoglutamicacidandglutamicacid(L-orD-isomers),respectively.IsoasparticacidandisoglutamicacidcanbeenzymaticallyconvertedtoAspandGlu(notshown).Morespecific,cross-linkingofWE14,achromograninADeamidationcanalsooccurnonenzymaticallyonasparagine7peptide,wasshowntoincreaseitsimmunogenicity.(Asn)andGln.NonenzymaticdeamidationatAsnoccursInterestingly,themajorHLAhaplotypethatconfersmainlythroughtheformationofanaspartylsuccinimidesusceptibilityforCD,DR3/DQ2,incombinationwiththeintermediate,whichisveryrapidlyhydrolyzedtoisoaspartic11haplotypeDR4/DQ8,alsocontributesthegreatestgeneticriskacidandasparticacid(Asp)(Scheme2B).Nonenzymatic8forT1D.BothDR3/DQ2andDR4/DQ8haveapreferencedeamidationatGlnoccursviaaglutarimideintermediateand12forbindingnegativelychargedanchorresiduesatvariousisthenhydrolyzedtoisoglutamicacidandGlu(Scheme2C).positions.DeamidationinducesanegativelychargedsideIsoasparticacidandisoglutamicacidcanbeconvertedtoAspchain,explainingthelinkbetweenHLAanddiseaseandGlu,respectively(notshown).Formationofthesix-susceptibilityandtheincreasedimmunogenicityofdeamidatedmember-ringglutarimideintermediateislessfavoredthanthe4,9,10peptidescomparedtotheirnativecounterparts.succinimideintermediateforAsn,causingnonenzymaticGlnEnzymaticdeamidationofGlnbyTGMenzymesinvolvesdeamidationtooccuratamuchslowerratethanAsn13thethiolgroupfromacysteineresidueintheactivesiteofthedeamidation.6enzyme.Theamide-groupofaGlnresidueonthesurfaceofaToidentifydeamidatedaminoacid(AA)residuesinproteinisattackedbythethiolgroup,producingathioesterproteins,liquidchromatography−tandemmassspectrometryintermediateandreleasingammonia.Thisacyl-enzyme(LC−MS/MS)isthemethodofchoice.However,asintermediateisthenhydrolyzed,resultinginthenetconversionnonenzymaticdeamidationsonAsn,andtoaminorextentoftheGlnresidueintoaGluresidue(Scheme2A).onGln,maybeintroducedinvitroduringsamplepreparation1406https://dx.doi.org/10.1021/acs.jproteome.0c00801J.ProteomeRes.2021,20,1405−1414

2JournalofProteomeResearchpubs.acs.org/jprTechnicalNote14forLC−MS/MS,theaccuratedetectionofinvivoTTAATTGCATTCAGCCCAGAGA-3′;TGM2forward:5′-deamidationsismarkedlyhampered.HereweaimedtoCTAAGAGTGTGGGCCGTGAT-3′andTGM2reverse:5′-optimizeamethodforimprovedvalidationofdeamidatedGCCAGTTTGTTCAGGTGGTT-3′.Primersofhousekeep-proteins,beingpotentialneoantigensinautoimmunediseases,inggenesusedfornormalizationwereActinforward:5′-byreducingtheoccurrenceofartifactualinvitrodeamidationAGAGGGAAATCGTGCGTGAC-3′;Actinreverse:5′-duringsamplepreparation.WereportthattrypsindigestionforCAATAGTGATGACCTGGCCGT-3′;RPL27forward:5′-90minatapHofeightreducesthenumberofinvitroAsnGTCGAGATGGGCAAGTTCAT-3′;RPL27reverse:5′-deamidationssubstantially,evidencedbydecreasedincorpo-TTCTTCACGATGACGGCTTT-3′;HPRTforward:5′-rationof18O,therebyfacilitatingthedetectionandverificationTGGCCATCTGCCTAGTAAAGC-3′andHPRTreverse:ofinvivodeamidations.Wealsopointtothefactthatalarge5′-GGCTCATAGTGCAAATCAAAAGTC-3′.Therelativepercentageofidentifieddeamidationswasassignedwrongly,foldgeneexpressionwascalculatedusingthedelta−deltaCtnamely28%and45%ofAsnandGlndeamidatedpeptidesmethod.withahighqualityMS2qualityrespectively,andprovideTGM2ActivityAssaydetailedinformationonhowtoverifydeamidationsbyTheactivityoftheTGM2enzymewasdeterminedwiththemanuallyinspectingMS2spectra,takingintoaccountthetissuetransglutaminasemicroassaykit(Zedira),accordingtopresenceofmultipleAsnand/orGlnresiduesandthepresence13themanufacturer’sinstructions.ofCisotopes.Asproofofprinciple,weshowthatinsulin-producingMIN6β-cellsinduceenzymaticdeamidationwhenOrbitrapLC−MS/MSexposedtocytokines,evidencedbyincreasedactivityoftissueMIN6cellswerelysed,reduced,alkylatedandproteintransglutaminaseortransglutaminase2(TGM2)paralleledbyprecipitatedaspreviouslydescribed.16Briefly,MIN6cellsincreasedlevelsofGlndeamidation.Strikingly,alsonon-werelysedin7mol/Lurea,2mol/Lthiourea,4%weightforenzymaticinvivooccurringdeamidationsonAsnareincreasedvolume3-(3-cholamidopropyl)dimethylammonio-1-propane-incytokine-exposedMIN6cells.Thesefindingsaddstrengthsulfonate,40mmol/LTrisbase,1%weightforvolumetotheexistingevidencethatdeamidationisoccurringindithiothreitol(DTT),andamixtureofproteaseinhibitorsstressedβ-cellproteinsandthatdeamidatedpeptidescan(cOmpleteproteaseinhibitor;RocheDiagnostics).CelllysatesgenerateanautoimmuneresponseinT1D.weredialyzedagainstMilli-Qwaterwiththeminidialysiskit,1kDacutoff(GEHealthcareGE80-6483-94).Celllysateswere■EXPERIMENTALPROCEDURESreducedin5mmol/LDTTfor30minat37°Cfollowedbyalkylationwith25mmol/Liodoacetamide(IAA)andCellCultureandTreatmentquenchingofexcessIAAwith25mmol/LDTT,bothbyMurineMIN6cells,agiftfromDr.Miyazaki(Osakaincubatingfor30minat37°Cinthedark.ProteinUniversity,Osaka,Japan),wereculturedinDMEM(In-precipitationwasperformedusingtheWessel-Flüggemeth-vitrogen)containing15%fetalcalfserum,100U/mLod.17Digestionwasperformedwithmodifiedtrypsin(Pierce)penicillin,100mg/mLstreptomycin,and70mmol/Lβ-in200mmol/Lammoniumbicarbonate(pH8)orammoniummercaptoethanol.Afterplating(8000cells/96-well,0.2×106acetate(pH6and5)(40μLfinalvolume,trypsin/proteincells/24-welland0.8×106cells/6-well),cellswereincubatedratio1:20(w/w))fortheindicateddurationinthepresenceofforatleast72hat37°Cbeforegoinginexperiments.MIN65%acetonitrile(ACN)and0.01%ProteaseMAX(Promega).cellswereexposedtorecombinantmouseIFN-γ(500U/mL;SolventevaporatedpeptidemixturesweresubjectedtoPeproTech),recombinantmouseTNF-α(1000U/mL;desaltingwithC18ZipTippipettips(Millipore)andsolventPeproTech),andrecombinanthumanIL-1β(10U/mL;evaporatedthereaftertoimprovestorage.Rightbeforeloading,R&DSystems)for14h.thedigestedsamplesweredissolvedin12μLloadingbufferCellDeathAssays(0.1%formicacid(FA)and5%ACN),ofwhich5μLwasinjectedandseparatedonanUltimate3000UPLCsystemThepercentageofapoptoticandlivingcellswasassessedby(Dionex,ThermoScientific)equippedwithanAcclaimcellcounting.MIN6cells(8000cells)wereexposedtoPepMap100precolumn(C18,particlesize3μm,poresizecytokinesfor14h,afterwhichtheywereincubatedfor10min100Å,diameter75μm,length20mm,ThermoScientific)andwithpropidiumiodide(4μg/mL)andHoechstHO342(20aC18PepMapRSLC(particlesize2μm,poresize100Å,μg/mL).Aminimumof500cellswerecountedblindineachdiameter50μm,length150mm,ThermoScientific)usingaexperimentalconditionwithaNikonEclipseTImicroscope.lineargradient(300nL/min).ThecompositionofbufferAisRNAExtractionandQuantitativeRT-PCRMilli-Qwatercontaining0.1%FA.ThecompositionofbufferTotalRNAwasextractedfrom0.2×106MIN6cells,usingtheBisMilli-Qwatercontaining0.08%FAand80%ACN.TheHighPureRNAIsolationKit(Roche)and500ngcDNAwasfractionofbufferBincreasedfrom0to4%in3min,from4−madeusingoligo-d(T)andsuperscriptIIreversetranscriptase10%in12min,from10−35%in20min,from35−65%in5(Invitrogen).QuantitativeRT-PCRwasperformedasmin,from65−95%in1min,andstayedat95%for10min.15previouslydescribed.Inshort,theamplificationreactionThefractionofbufferBdecreasedfrom95−5%in1minandwasperformedcontaining4pmolprimers,0.2μLcDNAand5stayedat5%for10min.TheQExactiveOrbitrapmassμLFastSYBRGreenMasterMix(AppliedBiosystems).spectrometer(ThermoScientific)wasoperatedinpositiveionPrimersusedwereCHOPforward:5′-TCTCATCCCC-modewithananosprayvoltageof2.1kVandasourceAGGAAACGAA-3′;CHOPreverse:5′-ATCTGGAGAG-temperatureof250°C.PierceLTQVelosESIpositiveionCGAGGGCTTT-3′;ATF4forward:5′-TCTGGAGGTG-calibrationmix(88323,ThermoScientific)wasusedasanGCCAAGCA-3′;ATF4reverse:5′-TCCAATCTGTCCCGG-externalcalibrant.Theinstrumentwasoperatedindata-AAAAG-3′;DP5forward:5′-dependentacquisitionmodewithasurveyMSscanataGGAGACCAGATGCCGTGAAA-3′;DP5reverse:5′-resolutionof70000(fwhmatm/z200)forthemassrangeof1407https://dx.doi.org/10.1021/acs.jproteome.0c00801J.ProteomeRes.2021,20,1405−1414

3JournalofProteomeResearchpubs.acs.org/jprTechnicalNotem/z400−1600forprecursorions,followedbyMS/MSscansexperimentally,i.e.,shorteningdigestiontimeandreducingofthetoptenmostintensepeakswith+2,+3,+4,and+5pH,resultedinanevenmorepronounceddecreaseofchargedionsaboveathresholdioncountof1e+6at17,500identifieduniquepeptides,rangingfrom4411±949downresolutionusingnormalizedcollisionenergyof25eVwithanto1403±878uniquepeptides,thelatterbeinga5.2-foldisolationwindowof3.0m/z(1.5m/zoneithersideofthereductioncomparedtostandarddigestionconditions(Figuremonoisotopicmass),apextriggerof5−15sanddynamic1A).Nexttothenumberofuniquepeptidesidentified,theexclusionof10s.AlldatawereacquiredwithXcalibur3.1.66.10software(ThermoScientific).Peptideswereidenti-fiedbyMascot(MatrixScience)usingSwissProt(Musmusculus,169779entries,January2018)asadatabasethroughProteomeDiscoverer2.2,incorporatingPercolatorforpeptidevalidation.Oxidation(M),deamidation(N/Q),anddeami-dation(R)(referringtocitrullination)wereincludedasvariablemodifications,andcarbamidomethylation(C)wasincludedasafixedmodification.Twomissedcleavageswereallowed,peptidetolerancewassetat5ppm,andMS/MStoleranceat20mmu.OrbitrapLC−MS/MSinH18O2ThesolventofMIN6celllysateswasevaporatedwithaSpeedVactoremoveall16Owater.Reduction,alkylation,proteinprecipitationanddigestionwasperformedin18O-labeledwater(97atom%18O,Sigma)asdescribedabove.C-terminalincorporationof18Oatomsanddeamidationbytheincorporationof18Oatomswereaddedasvariablemodifications.StatisticalAnalysisAlldatawereanalyzedusingGraphPadPrism8(GraphPad,LaJolla,CA).Statisticaltestsusedwerethepairedttestandtheone-wayANOVAwithDunnett’scorrectionformultipletesting.NormaldistributionofthedatawastestedwiththeShapiro−Wilktest.StatisticalanalysisofRT-qPCRdatawasperformedonΔCtvalues,analysisofTGM2activitydataonFigure1.Invitroartifactualdeamidationsdecreasewithshorterabsolutevalues.Unlessindicated,thedataisnotsignificant.*pdigestiontimeandlowerpH.(A)Totalnumberofuniquepeptides<0.05,**p<0.01,***p<0.001and****p<0.0001.identifiedafterdigestionatdifferentpH’s(8,6,and5)andduringDatadifferenttimes(overnight(ON),90and30min).(B−D)AmountofidentifieddeamidationsaccordingtotheMascotsearchenginebasedThemassspectrometryproteomicsdatahavebeendeposited18identificationandProteomeDiscoverervalidatedinthevarioustotheProteomeXchangeConsortiumviathePRIDEpartnerconditions(B)ordeamidationsonAsn(C)andGln(D)alone.repositorywiththedatasetidentifierPXD020301.Resultsareshownasmean±SD,n=3independentexperiments.Shapiro−Wilktestfornormalityandone-wayANOVAwith■RESULTSDunnett’scorrectionformultiplecomparisons,*p<0.05,**p<0.01,****p<0.0001.TheLevelofinVitroGeneratedNonenzymaticDeamidationsIsInfluencedbypHandDurationofTrypsinDigestionnumberofuniqueproteinsandtheaverageproteincoverageTobeabletoaccuratelystudyinvivogenerateddeamidations,werealsoevaluatedasasecondmeasureforevaluatingeitherenzymaticornonenzymatic,itisimportanttominimizedigestionefficiency(SupplementaryFigureS1).BoththethelevelofinvitrogenerateddeamidationsduringLC−MS/numberofuniqueproteinsidentifiedandtheaverageproteinMSsamplepreparation,astheyhampertheanalysisofthedatacoveragedecreasedtoasimilarextendastheuniquepeptidesset.SuchartifactualdeamidationsareoptimallyformedatpH8identifiedinthevariousconditionscomparedtothestandard14,19inanaqueousenvironment.Forthis,wepreparedMIN6trypsindigestioncondition,withaslightlybetteroutcomecelllysatesforLC−MS/MSundervariousexperimentalhoweverfordigestionfor90minatapHofeightcomparedtodigestionconditions,andevaluateddigestionefficiencyandtheotherconditions(831±87proteinsidentifiedvs931±56levelofdeamidation.inONpH8;and18.83%±0.86%proteincoveragevs20.42%Trypsindigestioninstandardconditions(overnight(ON),±0.23%inONpH8,respectively).pH8)resultedinthedetectionof7293±775uniqueLevelsofdeamidationinallthedigestionconditionswerepeptides.Shorteningthedigestiontimeto90and30minsignificantlyreducedcomparedtostandarddigestioncon-resultedinatime-dependentdecreaseinthenumberofditions,i.e.,rangingfrom43to64%reduction(p<0.0001forpeptidesidentified,with6064±880and5258±604uniqueall,Figure1BandSupplementaryTableS1).Ofnote,thepeptides,respectively.PerformingdigestionONbutreducingslightlyhigherlevelsinthenumberofdeamidationsinsamplesthepHto6and5,resultedinanotherdecreaseinidentifiedwithadigestionfor30minatpHsixorfiveismostprobablyuniquepeptides,with5217±1725and4919±1303attributabletoahighernumberoffalsepositives,duetotheidentifications,respectively.Combiningbothparameterslowoverallnumberofuniquepeptidesidentified.Deamida-1408https://dx.doi.org/10.1021/acs.jproteome.0c00801J.ProteomeRes.2021,20,1405−1414

4JournalofProteomeResearchpubs.acs.org/jprTechnicalNotetionswerefurtherclassifiedinAsndeamidationandGlnreductionindigestiontimeresultsinamajorreductionofindeamidation(Figure1C,DandSupplementaryTableS1).vitrogeneratedartifactualdeamidationonAsn,occurringWhilelevelsofGlndeamidationremainedsimilar,theduringsamplepreparation.Importantly,thisalsoindicatesthatreductioninthenumberoftotaldeamidationswasentirelyinvivogenerateddeamidationsarenotexclusivelyonGln,asattributabletothedecreaseinAsndeamidations(p<0.0001vsapproximately50%oftheinvivodeamidationscanbestandarddigestionconditionsforall),suggestingthatinvitroallocatedtochemicaldeamidationonAsn.artifactualdeamidationsonAsnwerelargelyreducedbyashorteneddigestiontimeandreducedpH.OnthebasisofManualInspectionofMS2SpectraofDeamidatedtheseresults,adigestionof90minatpH8waschosenasthePeptidesIsCrucialtoMinimizeFalsePositivesmostoptimalone,consideringthereasonablyhighefficiencyofBecausethedifferenceinmassbetweena13Cisotopeofadigestion(asindicatedbyamountofuniquepeptidesandpeptide(1.00335Dadifferenceinmassofisotopicpeakswithproteinsidentifiedandaverageproteincoverage).themonoisotopicpeak,Scheme3A)andadeamidatedversionInVitroArtifactualDeamidationsOccurMainlyonAsn,ofthepeptide(0.98402DadifferenceinmasswiththenativewhereasinVivoDeamidationsOccurBothonAsnandGlnpeptide)isonly19.34mDa(fullversusdashedlinesinSchemeTofurtherstrengthenandextendtheabovefindings,wenext3B),amassaccuracyof5ppmwasused.However,incorrectperformedproteomicssamplepreparationinthepresenceof1818monoisotopicionselectionduringdataacquisitionmaystillH2O.TheincorporationofOduringdeamidationresultsinanextraincreaseof2.00Da(twoextraneutronsfromthe18Ooccur,producingfalsepositiveidentificationsofdeamidatedinthecarboxylicacidgroupbuiltinduringthedeamidation)ontopofthe0.984Daincreaseofthedeamidation.ThisScheme3.VisualizedProcedureforVerificationofaallowstounambiguouslydistinguishbetweeninvitroDeamidationsinMS2Spectragenerateddeamidations(increaseof2.984Davstheunmodifiedpeptide)andinvivogenerateddeamidations(increaseof0.984Davstheunmodifiedpeptide).Todistinguishbetweeninvitroandinvivogenerateddeamidations,MIN6celllysateswerepreparedforLC−MS/MSinthepresenceofH18O,comparingONand90min2trypsindigestion,bothatpH8.Shorteningthedigestiontimeresultedinareductionininvitroartifactualdeamidation,with18Oincorporated,by70%(p<0.01,n=6)(Figure2A).TheFigure2.InvitroartifactualdeamidationsoccurmainlyonAsnwhereasinvivodeamidationsoccurbothonAsnandGln.18Olabeledinvitro(A)andunlabeled(16O)invivo(B)deamidationsonasparagine(Asn)andglutamine(Gln)afteranovernight(ON)or90mindigestion.Dataareshownasmean±SDfordeamidationsonAsnandGlnseparate.Shapiro−Wilktestfornormalityandpairedta(A)SchematicMS2spectrumofanunmodifiedpeptideconsistingoftestforthetotalofAsnandGln,**p<0.01comparedtoON,n=6fourAA.(B)MS2spectrumofthesamepeptide,deamidatedonGlnindependentexperiments.(Q)atposition3.Startingfromy2,themonoisotopicandtheisotopicpeaksareshiftedwith0.984Da.(C)SchematicMS2spectrumofthevastmajorityoftheseinvitrogenerateddeamidationswereonisotopeoftheunmodifiedpeptide,incorrectlyassignedasbeingAsnresidues,withonly16%occurringonGln,irrespectiveofdeamidatedonGln.Startingfromy2,thefirstisotopesareassignedasdigestiontime(Figure2AandSupplementaryTableS2).Thebeingthemonoisotopicpeaksofthedeamidatedpeptide,withthetotallevelofinvivodeamidation,determinedasthosewithoutdifferencesinmassbeing1.00Dainsteadof0.984Da.The18Oincorporated,beingnonenzymaticAsnandGlnmonoisotopicpeakoftheisotopeispresent,butnotassigned.(D)SchematicMS2spectrumofadeamidatedpeptide,withthedeamidationaswellasenzymaticGlndeamidation,wereasdeamidatedresidueincorrectlyassigned.Aty2,thefirstisotopeisexpectedunalteredupondecreaseofdigestiontimeandassigned,incorrectlyindicatingadeamidationatGln.Startingfromy,3occurredtoasimilarextentonAsnandGln(51%and49%themonoisotopicpeakisassignedcorrectly,meaningthatAsn(N)isrespectively)(Figure2BandSupplementaryTableS2).ThesedeamidatedinsteadofGln.FullpeaksarepresentintheMS2datastrengthenthefindingsaboveanddemonstratethataspectrum;dashedpeaksarenotpresentintheMS2spectrum.1409https://dx.doi.org/10.1021/acs.jproteome.0c00801J.ProteomeRes.2021,20,1405−1414

5JournalofProteomeResearchpubs.acs.org/jprTechnicalNotepeptides,evenwiththeuseofveryaccuratemassspectrometers.Here,wefirstevaluatedtheaccuracyoftheassigneddeamidatedpeptidesbymanualinspectionoftheMS2spectraforpossiblemisidentificationduetoincorrectassignmentofanisotopicpeakasadeamidation.TheprocedureisoutlinedinScheme3A−C,showingatheoreticalexampleofa4AApeptide,withadeamidatedGlnonposition3.Weconsiderthefragmentationspectrumoftheunmodifiedpeptideasareference.Startingfromy2,theyionscontainthedeamidatedGln.Alltheyionpeaksfromy2onwardshouldthusbeincreasedwith0.984Dacomparedtotheunmodifiedpeptide(Scheme3A,B).IncaseofincorrectassignmentattheFigure3.Distributionofcorrectandwrongfullyassignedprecursorlevel(deamidatedpeptideversusisotopeof13deamidations.Percentagesofhigh-qualitycorrectassignedGln(A)unmodifiedpeptide),startingfromy2theCisotopicpeaksandAsn(B)deamidations,deamidationswrongfullyassignedduringareassignedasmonoisotopicpeaksofthedeamidatedpeptidedataacquisition,isotopesthatwereassignedasdeamidationdueto(Scheme3C).Thedifferenceinmassbetweenthewrongfullyincorrectmonoisotopicselectionattheprecursorlevel,andpeptidesassignedpeaksandthetruemonoisotopicpeaksoftheyionsthatintheunmodifiedformbelongtoadifferentprotein.Dataareoftheunmodifiedpeptidethatwillalsobepresentinthiscase,shownasmean±SD,n=4independentexperiments.is1.00Da.Second,weevaluatedtheMS2spectramanuallyforpossiblewrongassignmentofthecorrectdeamidatedresidueinishighlyrecommended,evenwhenusingaccuratemasspeptideswithmultipleGlnand/orAsnresidues.Scheme3Dspectrometersandastrictsettingof5ppmasinthecurrentshowssuchexampleofa4AApeptide,withAsnatposition2protocol.andGlnatposition3,inwhichAsnisdeamidated.Inthiscase,CytokinesInduceProteinDeamidationinMIN6Cells,they2fragmentationpeakdoesnotcontainthemodification,ParalleledbyIncreasedTGM2mRNAExpressionandbutthe13CisotopicpeakcouldbewrongfullyassignedasbeingEnzymeActivitythemonoisotopicpeakofay2ioncontainingGlndeamidation.Asproofofprinciple,weaimedtoevaluatetheoccurrenceofStartingfromy3on,themodificationispresentinbothGlnandAsndeamidationsinMIN6cellsexposedtoscenariosandthemonoisotopicpeaks(increasedwith0.984inflammatorystress.Tothisend,wefirstinvestigatedtheDacomparedtotheunmodifiedpeptide)oftheyionsareregulationofTGM2itself,byevaluatingitstranscriptionalcorrectlyassigned.Inthiscasethedeamidationiswronglyexpressionlevelsandactivityuponcytokineexposure.MIN6assignedtotheGlnresidueduringdataacquisition,insteadofcellswereexposedinvitrotoIL-1β,IFN-γ,andTNF-αfor14totheAsn.h.ThiscytokineexposureresultedinincreasedapoptosisThird,weconductedabasiclocalalignmentsearchtool(32.18vs5.67%incontrolMIN6cells,p<0.001,n=4)(BLAST)analysisonthemanuallyassigneddeamidated(Figure4A)andendoplasmicreticulum(ER)/mitochondrialpeptidesfortheexistenceofproteinsfromthesamefamilystress,asevidencedbyincreasedmRNAlevelsofC/EBPwithidenticalsequenceexceptforanAsntoAsporaGlntohomologousprotein(CHOP),activatingtranscriptionfactor4Gluchange.(ATF4)anddeathprotein5(DP5)(5.92-,1.66-,and7.48-ExcludingMS2spectrathatwereoftoolowquality(notfold,respectively,p<0.05vscontrolMIN6forATF4andp

6JournalofProteomeResearchpubs.acs.org/jprTechnicalNoteFigure4.CytokineexposureinMIN6cellsinducesTGM2mRNAandactivity.MIN6cellswereexposedto50U/mLhIL-1β,250U/mLmIFN-γ,and1000U/mLmTNF-αfor14h.Levelsofapoptosis(A)andtranscriptionlevelsofCHOP(B),ATF4(C),andDP5(D)areincreasedincytokine-exposedMIN6cells.RNAlevels(E)andproteinactivity(F)ofTGM2areincreasedincytokine-exposedMIN6cells.Dataareshownasmean±SD,n=4independentexperiments.Shapiro−Wilktestfornormalityandpairedttest,*p<0.05,**p<0.01,***p<0.001comparedtocontrol.14ofAsn-Glysequences.Tobeabletoprovideamoreaccurateprofileofrelevantinvivodeamidationsweheredeviatedfromstandardtrypsindigestionconditionstoreduceartifactualdeamidations.Digestiontime,pH,andtemperatureareknowntobethreemajorfactorscontrollingtherateofinvitrodeamidation20duringproteomicssamplepreparation.Toourknowledge,thecombinationofasubstantialreductionindigestiontimeandpHhasnotbeenevaluatedbefore.Differentprotocolshavebeenreported,showingcompletedigestionefficiency20,21after30minorwithloweringthepHto6,althoughthesewereperformedonasinglerecombinantproteinsample.Figure5.Deamidationlevelsincontrolandcytokine-exposedMIN6Furthermore,Haoetal.reportedareductioninnonenzymaticcells.PercentageoftotaluniquepeptidesofGln(A)andAsn(B)deamidationsincomplexsamplesbyONtrypsindigestion,by22deamidatedpeptidesincontrolandcytokine-exposedMIN6cells.loweringthepHto6,whichisinlinewithourpresentShapiro−Wilktestfornormalityandpairedttest,*p<0.05,n=5findings.Ofnote,inthisstudyureawasusedasadenaturantindependentexperiments.duringdigestion,andwethereforecannotfullycompareourfindingswiththeirfindings.However,evaluatingboththecleoproteins(hnRNPs)(hnRNPA1deamidatedonN171,numberoftotaluniquepeptideidentificationsaswellastotalhnRNPA3deamidatedonN192,hnRNPD0deamidatedonuniqueproteinidentificationsandaverageproteincoverage,inN73,hnRNPHdeamidatedonN38,andhnRNPUdeamidatedourhandsdigestionfor90minatpH8showedtobeslightlyonN57),proteindisulfideisomerases(PDIs)(PDIdeami-moreefficientthanONdigestionatpH6.datedonN109andN451andPDIA3onN199andN474),ArecentstudybyLinetal.suggesteda2htrypsindigestiontetraspanin7(deamidatedonQ145),andsecretogranin-1timewithanenzyme-to-substrateratioof1:10astheoptimal(deamidatedonQ594)(SupplementaryFigureS2).condition,asthisresultedintheleastartificialmodifications23withmoderatenonspecificcleavagesandmiscleavages.For■DISCUSSIONourexperiments,anenzyme-to-substrateratioof1:20wasusedInthisstudywedescribeanimprovedmethodfortheforalltestedconditionsand,althoughalowerenzyme-to-detectionofAsnandGlndeamidationsincomplexproteinsubstrateratiomayhaveresultedinahighernumberofuniquelysates,makinguseofmurineinsulin-producingMIN6β-cells.peptidesduringONdigestion,wepreferredtokeepthisThemethoddecreasesthegenerationofartifactualinvitroparameterconstantforevaluatingtheeffectsoftimeandpH.deamidationswith70%,resultinginamarkedlyimprovedInterestingly,ouroptimaldigestionmethodisverysimilarto23detectionoftherelevantinvivoAsnandGlndeamidations.themethodsuggestedbyLinetal.WealsopointtothecriticalimportanceofmanualTheexactimpactonAsnandGlndeamidations,afterONvsinvestigationofMS2spectraoftheidentifieddeamidated90minoftrypsindigestion,wasfurtherevaluatedindetailbypeptides.ThefindingsfurtherhighlightthatMIN6cellsareperformingthesamplepreparationworkflowinthepresenceofH18O,therebydistinguishinginvitrofrominvivopronetodeamidationuponinflammatorystress,withan2increasednumberofbothAsnandGlndeamidations,deamidationsbyanextraincreaseof2.00DaforeveryinparalleledbyanincreasedexpressionandactivityofTGM2.vitrodeamidatedresidue.Thisrevealedareductionof70%Inrecentyears,deamidatedpeptideshaveemergedasaartifactualdeamidationsbyshorteningthedigestiontimefromsourceofalteredself-peptidesthataretargetedbyautoanti-ONto90min.Irrespectiveofthedigestiontime,16%ofthose4,5couldbeattributedtoGlndeamidations,provingthatinvitrobodiesandautoreactiveT-cellsinT1D.ThemethodofchoiceforthedetectionofsuchdeamidatedpeptidesisdeamidationsonGlnarenotnegligible,andalsoforthoseLC−MS/MS.However,duringstandardtrypsindigestionshorteningofthedigestiontimeisimportant.conditions,nonenzymaticartifactualdeamidationtakesplaces.Deamidations(bothenzymaticandnonenzymatic)aremostThisoccursmainlyonAsn,butalsotoasmallextentonGln,atoftenonlytakingplacepartially,therebygeneratingbothavariousratesdependingontheaminoacidC-terminalofthenativeandadeamidatedpeptide.Asthedifferenceinmassdeamidatedresidue,withupto70−80%invitrodeamidationbetweena13Cisotopeofanativeunmodifiedpeptideandthe1411https://dx.doi.org/10.1021/acs.jproteome.0c00801J.ProteomeRes.2021,20,1405−1414

7JournalofProteomeResearchpubs.acs.org/jprTechnicalNotedeamidatedversionofthispeptideisonly19.34mDa,amassimportanttargetsforimmunereactivityindifferentauto-errorofonly5ppmwasallowedinthedatabasesearches.Inimmunediseasesandtheincreasedinterestinthesearchfortheory,usingthisstrictsetting,deamidatedpeptideswithanoveldeamidatedneo-epitopes,webelieveacarefulevaluationmassupto3868Da(19.34mDa/5ppm)shouldbematchedoftheMS2spectraoftheidentifiedpeptidesiscrucialbeforecorrectlybyMS/MSanalysissoftware.However,manualproceedingtowardfunctionalanalysesinadiseasesetting.Theanalysisshowedthatinabout30%ofthehigh-qualityMS2presentfindingtherebyopenstheroadfordiscoveryofnovelspectratheisotopeofapeptidewasstillwrongfullyassignedasmodifiedautoantigens,notonlyinT1D,butalsoinotherbeingtheputativedeamidatedversionofthispeptide,autoimmunediseases.indicatingthatdataacquisitiondidreturnaquitehighnumberoffalse-positiveidentificationsofdeamidatedpeptides.■ASSOCIATEDCONTENTDuetothehighsimilarityinsequenceofproteinsfromthe*sıSupportingInformationsamefamily,apeptidecanbeidentifiedasdeamidatedonAsnTheSupportingInformationisavailablefreeofchargeatorGln,althoughitisactuallytheunmodifiedpeptidefromahttps://pubs.acs.org/doi/10.1021/acs.jproteome.0c00801.homologousprotein,withAsporGluinitssequence.Inourdataset,suchmisidentificationsresultedin7%falsepositivelyFigureS1:Uniqueproteinsandaverageproteincoverageundervariousdigestionconditions;FigureS2:MS2allocateddeamidatedpeptides.Anovelapproach,recentlyspectraofpeptideswithincreaseddeamidationuponestablishedbyWangetal.,allowstoeliminatesuchpeptidescytokine-exposure(PDF)automatically,withouthavingtoconductingaBLASTanalysisonallidentifieddeamidationsmanually.24Thisdual-searchTableS1:Amountandfractionoftotaluniquepeptidesdeltascorestrategyalsoallowstheautomaticexclusionoflow-ofidentifiedAsnandGlndeamidationsundervariousqualityspectra(about56%ofallinitiallyassignedAsndigestionconditions;TableS2:Amountandfractionofdeamidationsand66%ofinitiallyassignedGlndeamidationstotaluniquepeptidesofidentifiedinvitroandinvivoinourdataset)and13Cisotopes(about20and30%ofhigh-AsnandGlndeamidations;TableS3:ConfirmedGlnqualityspectraassignedasAsnandGlndeamidationdeamidationsincontrolandcytokine-exposedMIN6respectively),therebygreatlyenhancingthethroughputofcells;TableS4:ConfirmedAsndeamidationsincontroltheanalysis.Oneimportantpoint,whichstillneedsmanualandcytokine-exposedMIN6cells;TableS5:SummaryinspectionoftheannotateddeamidatedpeptidesevenwhentableofpropertiesofconfirmedAsnandGlnusingthisdual-searchdeltascorestrategy,istheassignmentofdeamidationsincontrolandcytokine-exposedMIN6thedeamidationtothecorrectresidueincaseofpeptideswithcells(XLSX)multipleAsnand/orGlnresidues.Inourdataset,suchmanualinspectionrevealedawrongassignmentduringdataacquisition■AUTHORINFORMATIONin8.39%ofthehigh-qualityspectra,withAsndeamidationCorrespondingAuthorinsteadofthepredictedGlndeamidation.LutOverbergh−LaboratoryforClinicalandExperimentalAlthoughtheroleofdeamidationinT1Diswellestablished,Endocrinology,KULeuven,3000Leuven,Belgium;theunderlyingfactorsleadingtoactivationofTGM2,andorcid.org/0000-0001-7126-356X;consequentlyincreaseddeamidationofproteins,arefarfromPhone:+3216377466;Email:lutgart.overbergh@clear.Marréetal.showedthatexposureofmouseisletstothekuleuven.bechemicalERstressinducerthapsigargin,actingthroughblockingofcalciumtransportfromcytoplasmintoER,resultedAuthors3inanincreasedTGM2activity.HereweshowthatexposureAïshaCallebaut−LaboratoryforClinicalandExperimentalofmurineβ-cellstoinflammatorycytokines,amoreEndocrinology,KULeuven,3000Leuven,Belgiumphysiologicalstressor,resultedinincreasedTGM2activity,RitaDerua−LaboratoryofProteinPhosphorylationandparalleledbyanincreasedlevelofGlndeamidations.OurProteomicsandSyBioMa,KULeuven,3000Leuven,BelgiumfindingsalsopointtoanincreaseofAsndeamidationsbySaurabhVig−LaboratoryforClinicalandExperimentalexposuretocytokines.AlthoughtheunderlyingmechanismEndocrinology,KULeuven,3000Leuven,Belgiumneedsfurtherinvestigation,thissuggeststhatnotonlyThomasDelong−SkaggsSchoolofPharmacyanddeamidationonGln,butalsoonAsnmayplayaroleinPharmaceuticalSciences,UniversityofColoradoAnschutz,immunogenicityinT1D.OurdatasetofMIN6β-cellsrevealedAurora,Colorado80045,UnitedStatesseveralproteinsofinterestwithincreaseddeamidationafterChantalMathieu−LaboratoryforClinicalandExperimentalcytokineexposure,suchasAsndeamidationsonGRP78,Endocrinology,KULeuven,3000Leuven,BelgiumhnRNPs,PDIs,andGlndeamidationoftetraspanin-7andCompletecontactinformationisavailableat:secretogranin-1.Tetraspanin-7isatargetofautoantibodiesin25,26https://pubs.acs.org/10.1021/acs.jproteome.0c00801type1diabetes.Secretogranin-1isasecretorygranuleprotein.MembersofthePDIfamilyandGRP78areenzymesNotesimportantinthecorrectfoldingandmaturationofproteinsintheER.AutoantibodiesagainstGRP78areadiagnosticmarkerTheauthorsdeclarenocompetingfinancialinterest.27inrheumatoidarthritis(RA)andcitrullinatedGRP78isan28,16autoantigeninmurineandhumanT1D.Citrullinated■ACKNOWLEDGMENTS29hnRNPA3isanautoantigeninRA.TheauthorsthankElineDesagerandMartineGilisInconclusion,thepresentfindingsunderscorethe(KULeuven,Leuven,Belgium)forexpertassistanceandDr.importanceofbothGlnandAsndeamidationinstressedβ-SebastienCarpentier(SyBioMaMassSpectrometry,KUcellsandprovideanimprovedmethodtoidentifytheseLeuven)forhelpfuladvice.ChadijaCallebaut’shelpinthemodifications.Withdeamidatedpeptidesemergingasdesignoftheschemesisgreatlyappreciated.Thisworkwas1412https://dx.doi.org/10.1021/acs.jproteome.0c00801J.ProteomeRes.2021,20,1405−1414

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9JournalofProteomeResearchpubs.acs.org/jprTechnicalNoteTargetinRheumatoidArthritis.ArthritisRheum.2001,44(4),761−771.(28)Rondas,D.;Crevecoeur,I.;D̀’Hertog,W.;Ferreira,G.B.;Staes,A.;Garg,A.D.;Eizirik,D.L.;Agostinis,P.;Gevaert,K.;Overbergh,L.;Mathieu,C.CitrullinatedGlucose-RegulatedProtein78IsanAutoantigeninType1Diabetes.Diabetes2015,64(2),573−586.(29)Sohrabian,A.;Mathsson-Alm,L.;Hansson,M.;Knight,A.;Lysholm,J.;Cornillet,M.;Skriner,K.;Serre,G.;Larsson,A.;Weitoft,T.;Rönnelid,J.NumberofIndividualACPAReactivitiesinSynovialFluidImmuneComplexes,butNotSerumAnti-CCP2Levels,AssociatewithInflammationandJointDestructioninRheumatoidArthritis.Ann.Rheum.Dis.2018,77(9),1345−1353.1414https://dx.doi.org/10.1021/acs.jproteome.0c00801J.ProteomeRes.2021,20,1405−1414