磺化修饰结合离子交换色谱和生物质谱富集鉴定含组氨酸肽段

20093March2009ChineseJournalofChromatographyVo.l27No.2158~163*:,,.Te:l(010)80705055,Emai:[e-mail].:(No.20505018,20635010,20735005,20875101)(No.2007CB914104,2006AA02A308).:20081103曹冬1,2,周春喜2,张养军2,韩春光2,邓玉林1,钱小红2*(1.,100081;2.,102206):N,pH30,(SCX),,,N,,N,,,:;;;;;:O658:A:10008713(2009)02015806:SulfonationmodificationassistedenrichmentandidentificationofhistidinecontainingpeptidesbystrongcationexchangechromatographyandmassspectrometryCAODong1,2,ZHOUChunxi2,ZHANGYangjun2,HANChunguang2,DENGYulin1,QIANXiaohong2*(1.BeijingInstituteofTechnology,Beijing100081,China;2.StateKeyLaboratoryofProteomics,BeijingProteomeResearchCenter,BeijingInstituteofRadiationMedicine,Beijing102206,China)Abstract:BythesulfonationattheNterminalofpeptides,thechargestateofhistidinecontainingpeptidesisdifferentfromthatofotherpeptidesinpH30solution.Basedonthisdifference,anewmethodwasdevelopedtoisolateandidentifysulfonatedhistidinecontainingpeptidesfromtrypticdigestofproteinsbystrongcationexchange(SCX)chromatographyandmatrixassistedlaserdesorption/ionizationtandemtimeofflightmassspectrometry(MALDITOFMS/MS).Usingthestandardproteinscontaininghistidinesasthemode,lthemethodologywasevaluated.TheresultsshowthatsulfonatedhistidinecontainingpeptideswereefficientlyenrichedbySCX,andtheNterminalsulfonationofthepeptidessimplifiestheinterpretationoftheacquiredmassspectraandfacilitatesthesequencingofhistidinecontainingpeptidesbyproducingconsecutiveandpredominantionsinpositivemodeMS2spectra,whichisthoughttobetheresultofthechargeneutralizationofbionsbytheNterminalsulfonicacidgroup.Thediscriminationofbionsandyionscangreatlyenhancetheconfidenceinpeptideandsubsequentproteinidentification.Itisfeasibletoisolateandenrichthehistidinecontainingpeptidesbyusingthismethodwhichhasthepotentialapplicationsinproteomics.Keywords:strongcationexchangechromatography(SCX);matrixassistedlaserdesorption/ionizationtandemtimeofflightmassspectrometry(MALDITOFMS/MS);sulfonationmodification;enrichment;identification;histidinecontainingpeptides(ESI)(MALDI),,2,:,,,,[1]Yates[2](MudPIT),1484[3],105,104,,,,,,,N[4-7](signaturepeptideproteomicsmethods)[8](globalinternalstandardtechniques)[9](isotopicallycodedaffinitytags)[10],(IMAC)[11,12]Cu()[13]IMAC,,()[14]Regnier[15]IMAC,IMAC,,IMAC,85%,N,pH30,,N,,,[16,17],,,11.1(MALDITOFMS/MS)(4700ProteomicsAnalyzer,)P230,(kemptide,LRRASLG)4(CHCA)Sigma(DTT)PromegaO(IAA)(THF)AcrosOrganics,2(SACA)Aldrich,J.T.Baker,MilliporeMilliQA101.21.2.1蛋白质的还原烷基化及酶切02mol/L(pH80,6mol/L)1mg/mL01mL,2L10mmol/LDTT,371h,2L20mmol/LIAA,1h02mol/L6,150,371.2.2标准肽的磺化反应条件优化实验,8,1nmol,pH60,65,70,75,80,85,9095100L02mol/L,10nmol200nmolSACA(SACA10200)4h,,MSH956/(H956+H772)(,H956,H772)H954/(H954+H770)(,H95415927,H770)1.2.3肽段的胍基化及磺化与含组氨酸肽段的色谱富集及质谱鉴定pH1101mol/LO374h,pH8,20mmol/LSACA,1hBetancourt[18],,(5m,30nm(300),200mm46mm),(5m,20nm(200),200mm46mm,PolySULFOETHYLA,PolyLC),A(60%,1%)10min,;B(60%,1%05mol/LNaCl),[19],MALDITOFMS/MS,6LC18ZipTip(),6L(5mg/mLCHCA01%TFA50%),06L(01g),,1kV,(CID)4700ExplorerV20()22.11:,,N,,Beausoleil[20],pH27,()NC,2N,C,,,1Fig.1PrinciplefortheenrichmentandidentificationofsulfonatedhistidinecontainingpeptidesbySCXchromatographiccolumn(SwissProt460)11936,971%,183%(127143/694584),11,(527%)29,(817%),,,,(973%961%)[21],ICAT(isotopecodedaffinitytag)2.22,,,1602,:,,(2)2SACA20010,pH2,pHSACA(10):pH75,pH90SACA(200)pH,pH60~85,pH90(2)2pH60~75,pHN(pKa75),pH,,213Table1EnrichmenthistidinecontainingpeptidesinthreemodelproteinsModelproteinNumberoftrypticpeptides1)NumberofexpectedhistidinecontainingpeptidesMrIsolatedpeptides(netcharge2))Myoglobin1261946.6#LFTGHPETLEJ(+1)1603.8#HGTVVLTALGGILJ(+1)1727.6#HPGDFGADAQGAMTJ(+1)1789.8#VEADIAGHGQEVLIR(+1)2110.0#YLEFISDAIIHVLHSJ(+2)2020.9#GHHEAELJPLAQSHATJ(+4)1002.4#HJIPIJ(+2)951.3#FDJFJ(+1)Lactoglobulin1313)1020.4#ALPMHIR(+1)Lysozyme1011057.4#HGLDNYR(+1)831.3#JVFGR(+1)1558.6#CJGTDVQAWIR(+1)1)ThenumberoftheoreticaltrypticpeptideswithMr500.2)thenetchargeofpeptidesinpH30solution.3)Thehistidinecontainingpeptide(LSFNPTQLEEQCHI)isCterminaloflactoglobulin,anddoesnotcontainlysineorarginine,thenetchargeofthispeptideiszero,notenrichedbyourmethod.2pHFig.2EffectofpHonthesulfonationmodificationofpeptides200timesSACAexcesstokemptide:1.innegativemode;2.inpositivemode.10timesSACAexcesstokemptide:3.innegativemode;4.inpositivemode.2.3[22],,(3),,(14),,,,,,3;,3Fig.3SCXchromatogramofguanidinatedandsulfonatedtrypticpeptidesofmyoglobinExperimentalconditions:strongcationexchangechromatographiccolumn(5m,20nm(200),200mm46mm,PolySULFOETHYLA,PolyLC);mobilephases:A,60%MeOHcontaining1%aceticacid;B,60%MeOHcontaining1%aceticacidand05mol/LNaClsolution;flowrate:1mL/min;detectionwavelength:214nm.H.histidinecontainingpeptides;N.nonhistidinecontainingpeptides.161274(a)(b)Fig.4Massspectraofguanidinatedandsulfonatedtrypticpeptidesofmyoglobin(a)beforeSCXseparationand(b)afterSCXseparationinnegativemodeH.histidinecontainingpeptides;N.nonhistidinecontainingpeptides.*.missedcleavagesitescontainingpeptides.5Fig.5MS2spectrumofaguanidinatedandsulfonatedhistidinecontainingpeptidein