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()276200612JournalofCapitalNormalUniversity(NaturalScienceEdition)Vol.27,No.6Dec.20063(,100037),,,:,,,,.:Q753:20052092273(Proteinphosphorylation),(Post2translationalmodifications,PTMs),2050.,1/3[1].,2%500100[2].,,.,.1,:O2N2S2.O2,,;N2;;S2.:(1),(S2N2),(PTR)(HPr);(2),,A()();(3).232P.Lees2MillerAnderson[3]32P,.deCarvalho[4]A2,32P.,.,;,,,.©1994-2007ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.[5]ABI470,PTH.[6]O2,pmol3PTH2,,32.[7],LRRASLG(Kemptide)A,,,.[8]H3.(ICAT)[9],.[10](PhIAT),,.P.Goshe[10],H3PO4,,.,.Zhou[11],,,..3,:311[12],,,IMAC[13,14].,(Fe3+Ga3+),pH.,,IMACRP2HPLC.IMAC.312..,,[15].TilleySchofieldt[16]Western2blotting,.Pandey[17],(EGF)HeLaHeLa,MS7EGFEGF.,[18]Western.,Western.[19].,Gronborg[20]6.3.7,5,1,Western-blotting31344()2006©1994-2007ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.:Lewis[21]2D,.Iwafune[22]2D20S.314,..,,,.,,.,,,,.[23](HPLC2ESI2QITMS)2(2casine)(myelinbasicprotein,MBP)..Claverold[24]SDS2PAGEK2.,,22DHPLCPMS22DPAGE/MS,.,,22DProteoSepTM[25].(pI),2-D.,(HPCF),pI,pI(RP-HPLC).3152D2PP,,(TCL)[26]..,(MS).316,.STAT[27].[28]IL5HL60,STAT3,STAT3,.:STAT3,STAT3.4,Edman,S22S2,.Edman,Edman,,.,,:411MALDI2TOF2MS,,MALDI2TOF2MS.Larsen[29],,1pmol.412MALDI2TOF2MS,,.[30].,,546:©1994-2007ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.[31].413MALDI2TOF2MSMuller[32]MALDI2TOF2MS.,.John[33],,MALDI2TOF2MS,,..Ma[34]MALDI2TOF2MS.,.414.(CID),..,[35].,.415Covey[36],,MS/MSCIDH3PO4(98u).,.,H3PO4..416ESI[37].,.m/Z63m/Z79.,,.41731PHPLC(ICP2MS)31P[38].(LC2ESI2MS).31P2NMR,.418(ECD)(FTICR).,CZ.CID.,,[39].,Edman[40].:Dupont[41]Edman2,2NKEL,.5[42],,Ser,Thr,Tyr2OHLys2NH2,,(Thr,Tyr)Ser-PO2-3Lys-PO2-3,.,.,.[43],,..Cohen[44]IkB-p105,SCF2TrCPSCF2TrCP.,.,[45].64()2006©1994-2007ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.:,,,.,,,.,.,..,.,.:,.,..,,,.,,.[1]ZolnierowiczS,BollenM.proteinphosphorylationandproteinphosphatases[J].DePanne,Belgium,1999,Sep,12-24.EMBOJ,2000,19:483-488.[2]MannM,OngSE,GronborgM,etal.AnalysisofproteinphosphorylationusingmassSpectrometry:decipheringthephosphoproteome[J].TrendsBiotechnol,2002,20:261-268.[3]Lees2MillerSP,AndersonCW.Twohuman902kDaheatshockproteinsarephosphorylatedinvivoatconservedserinesthatarephosphorylatedinvitrobycaseinkinaseII[J].JBiolChem,1989,264(5):2431-2437.[4]DeCarvalhoMG,McCormarkAL,OlsonE,etal.C.Identificationofphosphorylationsitesofhuman852kDaphospholipaseA2expressedininsectcellsandpresentinhumanmonocytes[J].JBiolChem,1996,271(12):6987-6997.[5]MeyerHE,Hoffmann2PosorskeE,Donella2DeanaA,etal.Sequenceanalysisofphosphotyrosine2containingpeptides.MethodsEnzymoh1991,201:2062224.[6],,,.[J].,1997,29(4):364-370.[7],.[J].,2000,32(1):69-73.[8],,,.H3[J].,2002,29(6):471-475.[9]GygiSP,RistB,GerberSA,etal.Qutitativeanalysisofcomplexproteinmixturesusingisotope2codedaffinitytags[J].NatureBiotechnology,1999,17:994-999.[10]GosheMB,ConradsTP,PaniskoEA,etal.Phosphoproteinisotope2codedaffinitytagapproachforisolatingandquantitatingphosphopeptidesinproteome2wideanalyses[J].AnalChem,2001,73:2578-2586.[11]ZhouH,WattsJD,AchersoldR.Asystematicapproachtotheanalysisofproteinphosphorylation[J].NatureBiotechnology,2001,19:375-378.[12]MuszynskaG,AnderssonL,PorathJ.Selectiveadsorptionofphosphoproteinsongel2immobilizedferricchelate[J].Biochemistry,1986,25(22):6850-6853.[13]PosewitzMC,TempstP.Immobilizedgallium()affinitychromatographyofphosphopetides[J].AnalChem,1999,71:2883-2892.[14]StensballeA,AndersenS,JensenON.CharacterizationofphosphoproteinsfromelectrophoreticgelsbynanoscaleFe()746:©1994-2007ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.[J].Electrophoresis,2001,22:207-222.[15]ThomasPC,TimothyDV.Anenrichedlookattyrosinephosphorylation[J].NatureBiotechnology,2005,23:36-37.[16]TilleyKA,SchofieldtJD.RapidcommunicationdetectionofphosphotyrosineinthehighMrsubunitsofwheatglutenin[J].Journalofcerealscience,1995,22:17-19.[17]PandeyA,PodtelejnikovAV,BlagoevB,etal.Analysisofreceptorsignalingpathwaysbymassspectrometry:identificationofvav22asasubstrateoftheepidermalandplateletderivedgrowthfactorreceptors[J],ProcNatlAcadSciUSA,2000,97(1):179-184.[18],.Western[J].,1997,23(6):678-680.[19],,,.[J].,2001,11(2):111-113.[20]GronborgM,KristiansenTZ,StensballA,etal.Amassspectrometry2basedproteomicapproachforidentificationofSerinePthreonine2phosphorylatedproteinsbyenrichmentwithphosphor2specificantibodies:identificationofanovelprotein,frigg,asaproteinkinaseAsubstrate[J].MolCellProteomics,2002,1(7):517-527.[21]LewisTS,HuntJB,AvelineLD,etal.IdentificationofnovelMAPkinasepathwaysignalingtargetsbyfunctionalproteomicsandmassspectrometry[J].MolCell,2000,6:1343-1354.[22]IwafuneY,KawasakiH,HiranoH.Electrophoreticanalysisofphosphorylationoftheyeast20Sproteasome[J].Electrophoresis,2002,23:329-338.[23],,.[J].C,2000,30(4):421-427.[24]ClaverolS,Burlet2SchiltzO,GairinJE,etal.Characterizat