生物质谱技术在细胞生物学中的应用桑志红王红霞综述摘要基因组计

,.10340post-genomeera,(),,2,11m/z80ESIMALDI19NF-kB[e.g.46]CD95FAS/APO-12529VHL18P241336,,RENT49.,.,DNA:Betts1a,Wong45calnexin)C,Carr5N-O-.,E-.2DPAGE)),,2DPAGEPI2DPAGE,MS1DSDS-PAGE,1DSDS-PAGE80,LC-MS/MS.,1D2DPAGE35S,2%/40%V/V1550ul10ml10ml15%/25%25%14GenomicSolutionsLtd.,St.Neots,UKShevchenko50%/5%150%310min0.02%w/v215min.310min1%1-1.5h21min0.04%2%5%1%(2D~50ul)5min50ul10-15minSpeed-Vac~15min,50ul10mMDTT100mM,5630minDTT15minSpeedVac15min50ul55mM100mM20min100mM15min,100mM1:13730min50ul15minSpeed-ac,50mM25ug250ul0.1%(15ul)415ul100ul50mM,13ng/ul.20ul445-60min50mM(10-20ul)371.MALDI-TOF,PMF100ppM,MALDI-TOFMSSaccharomycescerevisiaeCaenorhabditiselegansMALDI-TOFPSDpmolMS/MSMALDI-TOFMALDI-TOF2~3MALDI-TOFp24Jensen145%.10mg/mla--4-/2-1:1v/v4:1[10mg/ml/2-1:1v/v]0.3ml0.3ml5%0.5ml5%~1ml/MALDI-TOFESI-MS/MSMS/MSCIDCID1ESI-MSN,abcC-xyz,czabCO(28Da)immoniumacyliumAcyliumm/zy,yESIWilmMann1D2D1ul1MS/MSCIDPO3-m/z79puller(SutterInstrumentsNovato,CA,USA)ProtanaA/S;NewObjectiveInc.,Cambridge,MA;Micromass,Manchester,UKLC-MS/MSHPLC,Q-TOF,Micromass,Manchester,UKMS/MS,75180um,.,MS/MS1Gb,.LC-MS/MSdenovosequencing)LC-MS/MS(unattendedoperation).121BeowulfclustersNRDBdbESTNRDBSWISSPROTGENPETPdbEST/NationalCentreforBiotechnologyInformation/EuropeanBioinformaticsInstituteNCBI/EBIdbEST2000243,569,075~80%dbESTESTsdbEST~2-5%dbEST20Neubauer25SpliceosomeESTESTPeptidesearchfmolpmolfmol,gG,.SDS-PAGEp24Ip2423~27KDa1,4,6,7,8,12,33,34,37,38,40(Cargospecifictransport)p24Ip241,34,382Erp1pAErp1p12%SDS-48KdErp1pBAMALDI-TOFMS0.1DaErp1pErp25pEmp24pCCMALDI-TOFMSErv25pEmp24pDm/z687.3MS/MS1372.6DaAIELDIESGAEARErp25pBSaccharomycescerevisiaeEmp24Erv25pp241,34EREmp24pErv25p1Emp24Erv25pCOPIIEmp24Erv25pCOPII1S.Cerevisiaep24622p24ERP1ERP2EMP24ERV25Emp24,Erv25p,Erv1pErv2pErv1pErp1p22100ODSepharosemlErppSepharose100mMTris-HCI,pH8.0,100mMNaCI,5mMEDTA,1%TritonX-100,TritonX-100TNET20mlSDS12%SDS-PAGEMALDI-TOFMSBMALDI-TOFMSMALDI-TOFMS100ppmPepSeaEmp24p50%2B2CErv25p51%Emp24Erv25pCnano-ESI-MS/MSEmp24Erv25pCD687.3DaMS/MSErv25pAIELDIESGAEARBErp1pErp1pErv25pEmp24LC-MS/MSABErp1pErp1pErppYTFCLSNNYGTSPKNMYTVSSTESRErp2pErppKVEITLEKp24ERP2ERP4Erp222Erp4pErp2pErp2pErp4pp2422p24p24p24p24p24Xfl1pXFL1ORFYKL002WESTESTXFL1XFL1ZZXFL1C27S.MunropZZHIS5PCR30XFL1CZZTEV28IgG-SepharoseTEVIgG-SepharoseSepharoseMann2pMann5p(30)17Xfl1p(30)Xfl1pCXfl1pDNAPCRASPA-27Sigma-AldrichCo.Xfl1p1000OD1.2Sephroplast10mMpH7.5150mMNaCI,1%TritonX-100,2mMEDTA,25mlIgG-SepharoseFastFlow(AmershamPharmaciaBiotech,UK)4C1ml4150ml10TEVLifeTechnologiesLtd.,Paisley,UK4C35ml2SDS12%SDS-PAGEXfl1pTEVTEVTEV3ALCMSMS/MSLC-MS3B15m/z735.3MS/MS3CSMSEATGLLAGMNRXfl1pXfl1p3Xfl1pATEV-A12%SDS-PAGEXfl1p(B)LC-MS/MSQ-TOFm/z735MS/MSCm/z735MS/MS1468.6DaSMSEATGLLAGMNRXfl1pE-E-E-EGFFcChinesehamsterovaryCHON-nESI-MS/MS4m/z932.82795.4DaFcEEQYNSTYRMS/MS4E-.QIMSm/z204Q1Q3m/z204[HexNac]+m/z204Q1m/z932.8MS/MSQ32MALDI-TOFMALDI-TOFMALDILC-MS/MSMascotSequest1LC-MS/MSMS/MSRigautetal.(Rigaut,G.,Shevchenko,A.,Rutz,B.,Wilm,M.,Mann,M.,andSraphin,B.,(1999)NatureBiotechnol.17,1030-1032)TAP