Chemical Depletion of Histidine-Containing Peptides Allows Identi fi cation of More Low-Abundance Methylation Sites from Proteome Sample

《Chemical Depletion of Histidine-Containing Peptides Allows Identi fi cation of More Low-Abundance Methylation Sites from Proteome Sample》由会员上传分享，免费在线阅读，更多相关内容在学术论文-天天文库。

pubs.acs.org/jprArticleChemicalDepletionofHistidine-ContainingPeptidesAllowsIdentificationofMoreLow-AbundanceMethylationSitesfromProteomeSamplesQiWang,ZhouxianLi,JiahuaZhou,YanWang,KeyunWang,HongqiangQin,andMingliangYe*CiteThis:J.ProteomeRes.2021,20,2497−2505ReadOnlineACCESSMetrics&MoreArticleRecommendations*sıSupportingInformationABSTRACT:Proteinmethylation,especiallythatoccursonarginineandlysineresidues,isoneofthemostimportantpost-translationalmodificationsinvolvedinvariouscellularprocessesincludingRNAsplicing,DNArepair,andsoforth.Systematicanalysisofproteinmethylationwouldfacilitatetheunderstandingofitsregulatorymechanisms.Strongcationchromatographyhasbeenusedtogloballyanalyzearginine/lysinemethylationattheproteomescalewithgoodperformance.However,theco-enrichedhistidine-containingpeptidesseverelyinterferewiththedetectionoflow-abundancemethylpep-tides.Here,wedevelopedanovelchemicalstrategywhichenabledalmostcompletedepletionofhistidine-containingpeptidesintheproteindigest,therebyresultingintheidentificationofmorelow-abundancearginine/lysinemethylpeptides.Totally,333arginineandlysinemethylationformsfrom207proteinswereidentifiedinthisstudy.Overall,thenumberofmethylationidentificationsincreasedabout50%byusingournewmethod.DataareavailableviaProteomeXchangewiththeidentifierPXD023845.KEYWORDS:methylproteome,depletionofhistidine-containingpeptides,argininemethylation,lysinemethylation,antibody-free■INTRODUCTIONmethylatedproteinsorpeptides.ImmunoaffinityenrichmentwastheprevalentmethodforeitherarginineorlysineProteinmethylationisanimportantpost-translational8−10methylatedpeptides.However,thehighpriceandpoormodification(PTM)andisinvolvedinalmostallessentialprocessesofcellularbiology.1,2Eighttypesofaminoacidbatch-to-batchreproducibilityoftheseantibodieslimiteditsresiduescouldbemethylated.Amongthem,arginineandapplication.Meanwhile,eachantibodycanonlyenrichonelysinearethemostfrequentlymethylatedresidues.3Intypeofmethylationform,andthepan-specificantibodytoDownloadedviaUNIVOFCONNECTICUTonMay16,2021at17:47:54(UTC).addition,differentmethylationformscouldoccurononeenrichallthesixmethylationformsonarginineandlysineresidue.Theguanidinegroupofargininecouldbemono-ordi-residuesisstilllacking.Adomain-basedstrategywasanother11,12methylated(MMAorDMA)byproteinargininemethyl-affinityenrichmentmethodformethylatedproteins.Seehttps://pubs.acs.org/sharingguidelinesforoptionsonhowtolegitimatelysharepublishedarticles.4transferases(PRMTs).AccordingtothelocationofattachedAlthoughthecostofdomainswasmuchlowerthanthatofmethylgroups,argininedimethylationisfurtherdividedintoantibodies,theperformanceofmethylationidentificationwastwotypes,thatis,asymmetricandsymmetricdimethylation.verypoor.Thethirdmethodisachromatography-basedone,Forlysinemethylation,atmostthreemethylgroupscouldbewhichiscost-effectiveandhasarelativelygoodperformanceattachedtotheepsilonamineoflysinebylysineformethylpeptideenrichment.13Becausetrypticmethylpep-methyltransferases(KMTs);therefore,lysinemethylationtidesarealwayshydrophilicandcarrypositivecharges,theycouldbeclassifiedasmono-,di-,ortri-methylation(Kme1,couldbeenrichedbybothhydrophilicinteractionliquid5Kme2,andKme3).Itwasreportedthatdysregulationofchromatographyandstrongcationexchange(SCX)chroma-methylationinarginineandlysinewasalwaysassociatedwithtography.14−16Unfortunately,theidentificationofmethylpep-6,7tumorigenesisandneurodegenerativedisease.However,themechanismisnotfullyelaboratedsofar.ComprehensiveanalysisofproteinmethylationwouldprovideinsightsintoitsReceived:December3,2020complexregulatorymechanisms.Published:March8,2021Largescaleanalysisofarginine/lysinemethylationremainschallengingduetothesmallalterationofphysicochemicalpropertiescausedbymethylationfortheprotein/peptide.Todate,threestrategieshavebeendevelopedtoenrich©2021AmericanChemicalSocietyhttps://dx.doi.org/10.1021/acs.jproteome.0c009762497J.ProteomeRes.2021,20,2497−2505

1JournalofProteomeResearchpubs.acs.org/jprArticleFigure1.Workflowofthereverseenrichmentstrategy.Peptideswith>2positivechargesincludingmethylpeptidesareenrichedfromthetrypticdigestbyusingSCXatalowpHandthenaresubjectedtocarbonylationandcapturingbyhydrazideresintodepletethehistidine-containingpeptidestoimprovetheidentificationofmethylpeptides.tideswasseverelyinterferedbyhistidine-containingpeptidescentrifugation,andpeptidesinthesupernatantwereanalyzed13whenSCXwasused.WehavedevelopedahighpHSCXbyMS.approachtodecreasetheinterferenceofhistidine-containingCellCulturepeptides,whichyieldedgoodperformanceforargininemethylationidentification.14ConsideringthebindingoflysineFormetaboliclabellingofJurkatTcellsbyL-methionine-13CD,RPMI1640mediumlackingmethylatedpeptidestoSCXatpH9isweak,wedevelopedamethyl-3L-methioninelowpH-SCXmethodinwhichimmobilizedmetalionaffinity(Gibco)wassupplementedwith10%dialyzedfetalbovineserum(Gibco),0.1mM13CD,and1%chromatographywithcopperions(Cu-IMAC)wasusedtoL-methionine-methyl-3depletehistidine-containingpeptides.16Unfortunately,histi-penicillinandstreptomycin.Cellsweregrownat37°Cinadine-containingpeptidescannotbecompletelydepleted.Thus,humidifiedatmospherewith5%CO2forsevencelldoublings.theSCX-basedenrichmentstrategyneedstobefurtherProteindigestusedtotestthelabellingefficiencywaspreparedimproved.fromthelabeledcellsbyusingthefrequently-usedFASP21Here,wedevelopedanewSCX-basedreverseenrichmentprotocol.Whenthelabellingefficiencywashigherthan98%,strategytoidentifymorelow-abundanceproteinmethylationthecellswerethenusedinsubsequentexperiments.sites.PeptideswithoverthreepositivechargesatalowpHCellLysisandProteinDigestionwereenrichedbySCXasbefore,butthehistidine-containingTo5×107JurkatTcells,500μLoflysisbuffer(6Mpeptidesweredepletedbyimidazolecarbonylation,followedguanidinehydrochloride,100mMTris-HCl,pH8.5),10μLofbycapturingbyhydrazideresin(Figure1andFigureS1,0.5MTCEP,and40μLof0.5MCAAwereadded.Thecarbonylationrepresentsaseriesofmodificationsintroducingcarbonylgroup(s)inbiologyresearch;17−19weusedacroleinresultingsuspensionwasheatedat100°Cfor5min.Then,itwascooledandsonicated.Thelysatewasheatedat100°Cfor(ACR)tointroducecarbonylgroupstohistidineresidues).5minagain.Afterthat,itwascooledandcentrifugedat8000gThisstrategyenabledalmostcompletedepletionofhistidine-for30min,andthesupernatantwascollected.Thecontainingpeptidesandresultedintheidentificationofmorelow-abundancemethylpeptidescomparedwiththeconven-concentrationofproteinswasmeasuredbytheBCAmethod.tionalSCXmethod.Then,lysiscontaining1mgproteinwasmixedwith1×volumeof4°C-waterand8×volumesof−20°C-acetone.The■mixturewasincubatedat−20°Cfor2h.TheprecipitationEXPERIMENTSECTIONwascollectedbycentrifugingat2000gfor15minandwashedEnrichmentofSyntheticMethylpeptidestwicewith−20°Cacetone(80%).PelletwasdriedinairandThreesyntheticpeptides[GGHFYSAK,GGNFSGR(MMA)-resuspendedin400μLof10mMTris-HCl(pH8.0)byGGFGGSR,andGGNFSGR(aDMA)GGFGGSR]weredi-sonication.methyl-labelledinthe“light”form,desalted,anddried.20Then,1mgproteinswereincubatedwithtrypsin(1:100)andlysCtheywereredissolvedinPBSbufferandmixedatamassratio(1:100)at37°Cfor16h.Then,enzymeswereremovedbyaof20:1:1(22μgpeptidesintotal).1MACRwasaddedtothe10kultrafiltrationtube,andthefiltermembranewaswashedmixturetoafinalconcentrationof100mM.Thepeptideswerewith50μLof10mMTris-HCl(pH8.0).ThefiltrateswereincubatedwithACRat37°Cfor1handdesaltedinthecombinedandmixedwith60μLofArgCbuffer(50mMTris-subsequentstep.Then,thepeptideswereredissolvedin100HCl,50mMdithiothreitol(DTT),and2mMethyl-μLofdepletionbuffer(100mMNaAc,150mMNaCl,pHenediaminetetraacetate,pH7.65)and6μLof500mM5.5).50μLofhydrazideresins(UltraLink,Thermo)wereCaCl2.Then,thetrypticpeptideswerefurtherdigestedbywashedwith200μLofdepletionbufferthreetimesandmixedArgC(1:50)at37°Cfor16h.DTTinthesolutionwaswiththepeptidesolution.Theresultingsuspensionwasquenchedbyadding6μLof500mMCAAandheatedat95incubatedat25°Cfor1h.Finally,theresinswereremovedby°Cfor5min.Then,thepeptidesweredimethyl-labelledby2498https://dx.doi.org/10.1021/acs.jproteome.0c00976J.ProteomeRes.2021,20,2497−2505

2JournalofProteomeResearchpubs.acs.org/jprArticleadding60μLof600mMNaCNBH3and60μLof4%CH2OPRIDEpartnerrepositorywiththedatasetidentifierandincubatingat25°Cfor1h.Finally,thepeptideswerePXD023845.desaltedbytheHLBcartridgeanddried.DataAnalysisMethylpeptideEnrichmentbytheReverseEnrichmentMaxQuant(v1.6.4.0)wasusedtosearchtherawfilesagainstStrategytheUniprothumandatabasecontaining20,361entries(JuneASCX-SPEcolumnwasmadebypacking200μLofSCX2018).Maximalmiscleavagesitesfortrypsin/Pweresetastwo.Carbamidomethyloncysteine,13CD-labelledmethionine,andbeads(PolarMC30-SP,Sepax).Thebeadswerewashedwith3300μLofelutionbuffer(5mMKH2PO4,500mMKCl,pH“light”dimethyl(C2H6−)onthepeptideN-terminusweresetasfixedmodifications;‘heavy’mono-anddi-methyl(13CD−7.2,30%ACN)threetimes.Then,thesaltwaswashedawayby3and13CD−)onarginineandtwodifferentdi-methyl300μLofloadingbuffer(5mMKH2PO4,pH2.7,30%ACN)26(13CDCH−and13CD−)andonetri-methyl(13CD)onsixtimes.Peptidesdigestedfrom1mgproteinswere332639redissolvedin900μLofloadingbufferandloadedintothelysineweresetasvariablemodifications.OtherdefaultSCX-SPEcolumn.Then,peptideswithonlyonebasicaminoparametersremainedunchanged.Finally,peptideswithaacidresiduewerewashedawayby300μLofwashingbuffer(5scorelowerthan40wereremovedinsubsequentanalysis.mMKH2PO4,40mMKCl,pH2.7,30%ACN)sixtimes.ThesequencemotifaroundmethylationsiteswasanalyzedFinally,theremainingpeptideswereelutedby300μLofbyWeblogo(weblogo.berkeley.edu/logo.cgi).GOanalysiswaselutionbufferthreetimes.ThesamplewasdriedanddesaltedachievedbyusingSTRING(https://string-db.org/).fordirectLC−MS/MSanalysis,orthesamplewasdriedandredissolvedin600μLofwaterforthedepletionofhistidine-■RESULTScontainingpeptides.Totheabovesolution,66μLof1MACRDesignoftheEnrichmentStrategywasadded,andthemixturewasincubatedat37°Cfor1h.BecausethemethylationonarginineandlysineresiduesThen,thepeptidesweredesalted,dried,andredissolvedin50inhibitsthecleavagebytrypsin,theresultantmethylatedμLofdepletionbuffer(100mMNaAc,150mMNaCl,pHtrypticpeptidesalwayscarrymorepositivechargesthanother5.5).30μLofhydrazideresin(UltraLink,Thermo)wastrypticpeptidesatanacidicpH.Thismakestheenrichmentofwashedwith200μLofdepletionbufferthreetimesandmixed13,14methylpeptidesbySCXchromatographypossible.How-withthepeptides.Then,itwasincubatedat25°Cfor1h.ever,miscleavagecouldalsooccurevenwhenthesitesarenotFinally,theresinwasfilteredbyahome-madefilterunitandmethylated.Thiswillgenerateadditionalpeptidescarryingwashedwith200μLofdepletionbuffer.Thehome-madefilterover+2chargesatanacidicpHastheyhavemultipleLys/ArgunitwasmadebypushingasmallpieceofaporousTeflondiskresidues.Meanwhile,thechargeofhistidine-containingintoa200μL-tip.Thefiltratesfortheflowthroughandpeptidesisalsoover+2atanacidicpH.Thesepeptideswashingwerecombined.Trifluoroacetyl(TFA)wasaddedintocouldseverelyinterferetheenrichmentofarginine/lysinethecombinedfiltratetoafinalvolumefractionof1%.2mgmethylatedpeptidesfromtheproteindigestbySCX.WeHLBbeadsweresuspendedin100μLofmethanol.Then,thedevelopedanenhanceddigestionstrategybyusingmultiplesuspensionliquidwasaddedintoanewhome-madefilterunit.proteases,thatis,trypsin,lys-C,andarg-C,whichdramaticallyAfterthemethanolwasfilteredout,thebeadswerewashed14decreasedtheoccurrenceofmiscleavage.WeappliedCu-with300μLof0.1%TFA.Thefiltratewasloadedontothe16IMACtodepletethehistidine-containingpeptides.Un-beads,andthebeadswerewashedwith100μLof0.1%TFA.fortunately,thehistidine-containingpeptidesonlyslightlyPeptidesabsorbedinthebeadswereelutedby150μLofbufferdecreasedfrom85to62%.Duetothepoorperformanceofthe(0.1%TFA,80%ACN).Finally,thesamplewasdriedanddepletion,thenumberofidentifiedmethylpeptidesfromthreeanalyzedbyliquidchromatography−massspectrometry(LC−MSreplicateswasnotincreased.Therefore,itisstillnecessaryMS)/MS.todevelopamoreeffectivemethodtodepletehistidine-MSAnalysiscontainingpeptides.ThesyntheticpeptidesweredetectedbyanABSciex5800ItiswellknownthatACRcouldreactwithahistidine22matrix-assistedlaserdesorptionionizationtime-of-flightresidue.Thisreactionwillintroduceonealdehydegroupto(MALDI-TOF)/TOFmassspectrometerwithapulsedNd/thehistidineresidue,whichmakesthedepletionofhistidine-YAGlaserat355nm.DHBwasusedastheMALDImatrix.containingpeptidesbyhydrazideresinpossible.However,Samplespreparedfromthecellswereredissolvedin0.1%TFAhistidineisnottheonlyresiduethatcouldreactwithACR.andmeasuredbyaQExactivemassspectrometer(ThermoCysteineandlysineresiduescouldalsoreactwithit.Scientific)equippedwithanUltimate3000RSLCnanosystemFortunately,afterproteinsareextractedfromcells,cysteine(Dionex).TheconcentrationofpeptideswasmeasuredbyaresidueswouldreactwithCAA.ThiscouldpreventtheNanoDropOne(ThermoScientific),and1μgpeptideswerereactionbetweencysteineandACR.Forlysineresidues,weloadedona5cm×150mmtrapcolumn(ReproSil-PuruseddimethyllabellingtodecreaseitsreactivitytoACR.After120C18,1.9μm)andseparatedbyusingalineargradientwithdimethyllabelling,aminogroupsinboththelysineresidueandaflowrateof550nL/minover120minina15cm×150mmN-terminuscannotreactwithACRanymore(FigureS2).Toanalyticalcolumn(ReproSil-Pur120C18,1.9μm).Inthemassfurtherprovethatmethylatedaminoacidresiduescannotreactspectrometer,MS1spectra(massrange300−1750Th)werewithACR,peptideswithKme1,Kme2,Kme3,MMA,ADMA,acquiredataresolutionof70,000at200Th.Top10ionswereandSDMAwerefirstsubjectedtodimethyllabellingandthenselectedforMS2scans,anda2ThisolationwindowwasusedincubatedwithACR.ItwasfoundthatallofthemethylatedforHCDfragmentation.MS2spectrawereacquiredataresiduescannotreactwithACR(FigureS2).Intheory,Kme1resolutionof35,000at200Th.isasecondaryaminewhichcouldreactwithACR,butitwasThemassspectrometryproteomicsdatahavebeendimethylatedafterdimethyllabelling,soitcannotfurtherreactdepositedtotheProteomeXchangeConsortiumviathewithACReither(whenthecellswerelabelledbyheavy2499https://dx.doi.org/10.1021/acs.jproteome.0c00976J.ProteomeRes.2021,20,2497−2505

3JournalofProteomeResearchpubs.acs.org/jprArticleFigure2.Depletionofhistidine-containingpeptidesfromthemixtureofthreesyntheticpeptides:(A)peptidesafterdimethyllabelling;(B)peptidesafterreactionwithACR;and(C)peptidesafterdepletionofhistidine-containingpeptidesbyhydrazideresin.methionine,themethylgroupinKme1wasisotopicallycarryingover2+charges,nearly80%ofpeptideshadatleastlabelled.Thus,Kme1wouldhaveoneheavymethylgrouponehistidineresidue.However,thispercentagewasdramat-duetothemetaboliclabellingandonelightmethylgroupdueicallydecreasedto<1%whenthehistidine-containingpeptidestothedimethyllabelling.ItcouldbedistinguishedfromKme2werecarbonylatedanddepletedbyhydrazidebeads.Evenwithtwoheavymethylgroups).Incomparison,thehistidinewhentheadditionalmodificationsinducedbyACRweretakenresidueistheonlyresiduethatcouldreactwithACR(Figureintoconsideration,thehistidine-containingpeptidesstillS2).However,theresultingproductwasnotpureasseveralaccountfornomorethan2%(FigureS3).Incomparison,peakswereobserved.Becauseofself-polymerizationofACR,therewerestill65%histidine-containingpeptidesevenafterthemassshiftinducedbythereactionwasnotonly56DabutthedepletionbyCu-IMAC.16alson×56Da.However,ifalloftheseproductshavealdehydeAfterthedepletionofthemaininterferencepeptides,wegroups,theywouldbedepletedbyhydrazidebeads.Toprovethenevaluatedtheperformanceofthismethodforthishypothesis,wemixedthehistidine-containingpeptidewithmethylpeptideidentification.Asexpected,thenumberoftwoabovementionedargininemethylatedpeptidesastheidentifiedmethylpeptidesincreasedfrom76to121inasinglesampletoevaluatethisdepletionmethod(Figure2A).runafterSCXenrichment(Figure3B),andthisnumberObviously,theoriginalpeakofthehistidine-containingpeptidefurtherincreasedto150whenthehistidine-containingdisappearedafterthereactionwithACR(Figure2B),butthepeptidesweredepleted.Theenrichmentspecificity,thatis,peaksforargininemethylatedpeptidesremainedasbefore.thepercentageofmethylpeptideidentifications,alsoincreasedFinally,hydrazidebeadswereusedtocapturehistidine-from1.3to3.2%(Figure3C).Intermsofidentificationcontainingpeptides.Thenewlyproducedadductpeakswerenumbers,theimprovementinducedbythedepletionwasnotalmostcompletelydisappearedinthespectrum(Figure2Candsobig.Thisispartlybecausethehistidine-containingTableS1).Meanwhile,peaksofmethylatedpeptidesmethylpeptideswerealsodepleted.Indeed,thenumberofdominatedthespectrum.Thisindicatedthatourstrategyhistidine-containingmethylpeptidesdecreasedfrom31tocouldefficientlydepletehistidine-containingpeptides.seveninasinglerunafterthedepletion.However,ifonlyMethylproteomicsAnalysisbytheNewStrategymethylpeptideswithouthistidinewereconsidered,thenumberToevaluatetheefficiencyofourstrategy,itwasappliedtoincreasedby58.9%byusingthisnewmethod.WethenenrichmethylpeptidesfromthetrypticdigestofthetotalcellcomparedtheidentificationsfromthreeMSreplicatesbeforelysateofJurkatTcells.Becausemethylproteomicsanalysisandafterthedepletion.Thenumberofmethylpeptidesalwayshasahighfalsediscoveryrate,23weusedthe‘heavy’increasedfrom196to263afterdepletion(Figure3D).Informofmethioninetometabolicallylabelcellstoimprovethecomparison,thisnumberwasnotincreasedbydepletionby16usingCu-IMAC,aswereportedbefore(FigureS4),16whichidentificationconfidence,asreportedbefore.Theproteinswereextractedfromthecellsanddigestedbytheenhancedconfirmedthebetterperformanceofthisnewmethod.digestionstrategydevelopedinourlaboratorytodecreasetheHowever,itwasstrangethattherewerehalfofoccurrenceofmiscleavage.Thepeptidesweredimethyl-methylpeptideswithouthistidinethatwerelostafterthelabelledtoblocktheprimaryaminesandthensubjectedtodepletion(Figure3E).Tofurtheranalyzethereasonofthelow-pHSCXenrichment.Asexpected,thepercentageofloss,wequantifiedmethylpeptidesbeforeandafterthepeptidescarryingover2+chargesincreasedfrom28.7todepletion.Asaresult,onlyasmallnumberofpeptideswere96.8%aftertheenrichment(Figure3A).Amongthepeptidesobservedtodecreaseinabundanceafterthedepletion(Figure2500https://dx.doi.org/10.1021/acs.jproteome.0c00976J.ProteomeRes.2021,20,2497−2505

4JournalofProteomeResearchpubs.acs.org/jprArticleFigure3.PerformanceofthereverseenrichmentstrategyfortheanalysisofmethylproteomefromJurkatTcells.(A)Distributionofdifferenttypesofpeptidesidentifiedaftertheanalysisofthetotalproteins,SCX-enrichedsample,andhistidine-containingpeptide-depletedsample.(B)Averagenumbersofidentifiedmethylpeptidesfromthreeruns.(C)Percentageofmethylpeptideidentifications.(D)Venndiagramofallidentifiedmethylpeptidesbeforeandafterdepletionofhistidine-containingpeptides.(E)Venndiagramofidentifiedmethylpeptideswithouthistidineresiduesbeforeandafterdepletionofhistidine-containingpeptides.TheerrorbarsinthisfigurerepresentthreestandarddeviationsforthreeMSreplicates.4A).ThenumbersofmethylpeptideswithincreasedandHowever,eventhoughsomemethylpeptideswerelost,unchangedabundancewerealmostthesame.Themethylpep-additionalidentifiedmethylpeptidesweremuchmorethantideswereclassifiedintotwogroupsaccordingtowhetherthethelostones.Thisisanewwaytodetectadditionallow-foldchangewasincreasedmorethantwotimesaftertheabundancemethylationsites.depletion.Obviously,thelengthsofmethylpeptidesinthetwoUnknownMethylationSitesIdentifiedbyThisEnrichmentgroupsweredifferent(Figure4B).ThepeptidesincreasedinStrategyabundancewerelongerthantheotherpeptides.Inotherwords,shortermethylpeptideswereeasiertobelostintheThemethylationformsonidentifiedmethylpeptideswereprocessofdepletion.Thismaybebecausethepeptideswereanalyzed.Itwasfoundthatthenumberofargininemethylationlostinthetwostepsofdesaltingbecauseoftheweakerformsincreasedbyabout56%,whilethenumberoflysineretentionofshorterpeptidesonHLBbeads.However,bothmethylationformsshowedalmostnochangeafterdepletionofthedesaltingstepafterthereactionofcarbonylationtoremovehistidine-containingpeptides(Figure5A).ThisnewmethodACRandthedesaltingstepbeforeLC−MS/MSanalysisareshowsthebetterperformanceintheidentificationofargininenecessary,anditishardtofurtherimprovetheworkflow.methylation.Itshouldbenotedthateventhoughtheidentified2501https://dx.doi.org/10.1021/acs.jproteome.0c00976J.ProteomeRes.2021,20,2497−2505

5JournalofProteomeResearchpubs.acs.org/jprArticleFigure4.Quantificationofmethylpeptidesbeforeandafterdepletionofhistidine-containingpeptides.(A)Volcanoplotofmethylpeptidesbeforeandafterdepletionofhistidine-containingpeptides(n=3).(B)Boxplotofthelengthofmethylpeptideswithincreasedandnotincreasedabundanceafterthedepletion.Afoldchangeoftwowasselectedasthecriteriontoclassifythemethylpeptidesintotwogroups.Figure5.Analysisofidentifiedmethylationforms.(A)Averagenumbersofidentifiedmethylationformsfromdifferentsamplesfromthreeruns.(B)Venndiagramoflysinemethylationformsidentifiedbeforeandafterdepletionofhistidine-containingpeptides.(C)PercentageofknownmethylationformsinthePhosphoSitePlusdatasetforouridentifiedresults.(D)Venndiagramofargininemethylationformsidentifiedbeforeandafterthedepletion.9numberwasnotincreased,59additionallysinemethylationantibodieshasgoodperformance.However,theantibodyforformswereidentified(Figure5B).Alloftheidentifiedformslysinemethylatedpeptidesenrichmentwasnotgoodenoughaswerecomparedwiththeknownmethylationdatasetinfarasweknow.ThiscouldbeduetothehugedifferencePhosphoSitePlus.Itwasfoundthatahigherproportionofcausedbydifferentmotifsaroundarginine/lysinemethylationunknownlysinemethylationformswasidentifiedafterthesites.UnlikeotherPTMssuchasphosphorylation,thechangedepletion(Figure5C).Thisindicatedthatthedepletionwasofthephysicochemicalpropertyoflysineandarginineinducedhelpfulfortheidentificationoflow-abundancelysinebymethylationwastoosmalltobespecificallyrecognizedbymethylationforms.antibodies.Thus,themotifaroundmethylatedsiteswasvitaltoIncomparisonwiththehighpercentageofunknownlysinemethylpeptideenrichmentusingantibodies.Thesuccessofmethylationsitesidentified,morethanhalfofthearginineantibodiesfortheenrichmentofargininemethylatedpeptides24methylationsiteswerealreadyknown.ThisisbecausethewasbenefitedbythemotifsuchastheRGGbox.Several8immunoaffinityenrichmentbyusingargininemethylationargininemethylationantibodiesweredesignedbasedonthis.2502https://dx.doi.org/10.1021/acs.jproteome.0c00976J.ProteomeRes.2021,20,2497−2505

6JournalofProteomeResearchpubs.acs.org/jprArticleFigure6.BioinformaticsanalysisofidentifiedmethylproteomicsinJurkatTcells.(A)GOanalysisofargininemethylatedproteins.(B)GOanalysisoflysinemethylatedproteins.(C,D)Motifanalysisofarginineandlysinemethylationsites.Unfortunately,therewasalmostnolysinemotifdiscoveredasfromthreeMSreplicates.Especially,itshowedbetterfarasweknow.Thislimitedthedevelopmentofantibodiesforperformanceforidentificationofmethylpeptideswithoutthelysinemethylationanalysis.Thus,identificationoflysinehistidineresidue.Althoughpartofmethylpeptideswerelostmethylationismorechallenging,andournewenrichmentduringthedepletionstep,ityieldedmorelow-abundancestrategyallowsidentificationofmorenewlysinemethylationmethylpeptideidentifications.Asfarasweknow,arobustforms.methodformethylationidentificationcoveringallkindsofBioinformaticAnalysisoftheObtainedMethylproteomeformsonarginineandlysineisstilllacking.ThecombinationaluseofmultiplemethodscouldbeaneffectivewayforOverall,weidentified333methylationformscorrespondingtocomprehensiveanalysisofproteinmethylation.207proteinsinJurkatTcells(TableS2).GOanalysisofargininemethylatedproteinsindicatedthattheywereenriched■inregulationoftranscription(Figure6A),buttheenrichmentASSOCIATEDCONTENT*sıSupportingInformationwasnotveryobviousforlysinemethylation(Figure6B).ItwasreportedthatlysinemethylationparticipatesinalmostallTheSupportingInformationisavailablefreeofchargeat5https://pubs.acs.org/doi/10.1021/acs.jproteome.0c00976.essentialbiologicalprocesses.Ourresultsindicatedthecomplexityofproteinregulationbylysinemethylation.ThisIntensityofdifferentpeptidesinMALDIMSbeforeandcanalsobeevidencedbythemotifanalysis.Incomparisonafterdepletionofhistidine-containingpeptides;identi-withargininemethylationwithobviousmotifs,sequencesfiedmethylationformsinthisstudy(XLSX)aroundlysinemethylationsiteshavenofeature(Figure6C,D).Chemicalreactionsinthisstudy;MALDIMSanalysisofIngeneral,theresultsofGOanalysisforidentifiedmethylated24syntheticpeptideswithdifferentresiduesanddifferentproteinsweresimilartopreviousstudies.methylationformsbeforeandafterdimethyllabelingand■reactionwithACR;percentagesofhistidine-containingCONCLUSIONSpeptideswithdifferentmodificationsinallidentifiedAsoneofthesmallestPTMs,proteinmethylationposesahugepeptides;andcomparisonofthenumberofmethylpep-challengeforitsanalysis.Enrichmentofmethylpeptideswastidesidentifiedfromdifferentsamplesusingdifferentthekeystepforthesensitiveidentification.Althoughtheremethods(PDF)wereseveralmethodsavailable,eachmethodhasitslimitations.Developmentofanalternativeenrichmentstrategy■isstillnecessarytoidentifymoreunknownmethylationsites.AUTHORINFORMATIONInthisstudy,toimprovetheperformanceoftheSCX-basedCorrespondingAuthormethod,co-enrichedhistidine-containingpeptideswereMingliangYe−CASKeyLaboratoryofSeparationSciencescarbonylatedanddepletedbyhydrazideresin.ComparedforAnalyticalChemistry,NationalChromatographicR&AwithconventionalSCX-basedmethods,thisnewmethodCenter,DalianInstituteofChemicalPhysics,Chineseresultedina30%increaseformethylpeptideidentificationsAcademyofSciences,Dalian116023,China;Universityof2503https://dx.doi.org/10.1021/acs.jproteome.0c00976J.ProteomeRes.2021,20,2497−2505

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