应用nanoLCMSMS对人羊水的蛋白质组学分析

291()Vol.29No.120082JournalofJinanUniversity(NaturalScience)Feb.2008[]2007-11-26[](30600587);(51205066)[](1972-),,,,:.E-mail:[e-mail]/MS/MS1,1,2,1,1,1,2(1.,510632;2.,511400)[]nanoLC/MS/MS.,CibacronblueProteinAIgGs,,nanoLC/MS/MS,62.,22,4.nanoLC/MS/MS.[];;nanoLC/MS/MS[]R714.12;Q517[]A[]1000-9965(2008)01-0099-06ProteomeanalysisofhumanamnioticfluidbynanoLC/MS/MSJIYu2hua1,ZENGYao2yin1,ZHOUJian2,HEXian2hui1,XINFei2yue1,ZHAOJing2xian1,ZHUBin2(1.InstituteofTransplantationandImmunology,JinanUniversity,Guangzhou510632,China;2.DepartmentofGynecologyandObstetrics,HexianHospital,Guangzhou510632,China)[Abstract]Toestablishproteomeanalysismethodsforhumanamnioticfluid(AF)withnanoLC/MS/MSforsearchingspecificproteinexpressedinAF.TwospecimensofnormalAFwerecollectedbytrans2amnioticpuncture,albuminandIgGsweredepletedbyCibacronblueandproteinAadsorption,thentheproteinsamplesweredenatured,reduced,alkylated,digestedandanalyzedbynanoLC/MS/MS.TheproteinsidentifiedinthepresentstudywerecomparedwiththeproteinsalreadyidentifiedinAFandhu2manplasma.62proteinsidentifiedinthepresentstudy,amongwhich22werethenewfoundproteinsex2clusivelypresentedinAF,4ofthemhadalreadybeendescribedtobeplacentaorpregnancyspecific.Inthepresentstudyweestablishproteomeanalysismethodsforhumanamnioticfluid(AF)withnanoLC/MS/MSandfindseveralpossibleAFspecificproteincandidates.[Keywords]amnioticfluid;proteomics;nanoLC/MS/MS(amnioticfluid,AF),[1].,-fetoproteinprolactinhCG,(prematureruptureofftealmembranes,PROM),.,.nanoLC/MS/MSAF,,.11.1DTTTrisbase(promega);(Pierce);(Merck),(Fluka);Milli-Q(18M)YM30145m(Millipore);IgG(AurumTM)Brandford(Biorad);Marker().1.2(37)5mL,416000g15min,0.4m-70.,AurumTM400LIgG,,Brandford,SDS-PAGE.400g8mol/L50mmol/LTris-HCl(pH8.0),10mmol/LDTT371h,25mmol/L30min,100mmol/LTris-HCl(pH8.0)2mol/L,150,3716h.1.3NanoLC/MS/MS0.1%NanoLC/MS/MS.Agilent1100Nano2flowHPLCtrap,C18Vay2dac218MSTM(150m150mm,Grace),C18PepMapTM(300mid5mm,LCPackings),A(0.1%)B(0.1%),18L,,1L/min.5%B5min5%40%B150min40%80%B1min,80%B10min5%B26min.TOF(QSTARXL,ABI)ESI/MS/MS,,MS4001600m/z,MS3(10,+2+4)IDA(informationdependentacquire),,MS/MSIDA(informationdependentacquire),3.BSA.1.4ProID(ABI),Swiss-Prot,:MS0.15amu,MS/MS0.1amu,1,,1.3(95%).22.1Brandford115210mg/mL,50%70%IgGs.,,TCA/.CibacronblueproteinAIgGs,SDSPAGE(Mw66ku)(1).M:Marker(97.466.2433120.114.4ku)AF:;unbound:AF;bound:AFIgGs1AFSDS-PAGE2.2nanoLC/MS/MS,2DE/MS.2DE/MS,2DE,,pH.001()29(LC/MS/MS),2DE/MS.,[2].PROM5%,,,PROM.Vuadens[3]2DE/MSAF,AFperlecan(P98160)agrin(O00468).Nilsson[4]LC/FT-ICR/MS43,Michel[5]OGE/LC/MS/MS69,,LC/MS/MS,,Michel.nano-LC/MS/MS62(2,1),Michel[5]32,30,.Li[6],;Anderson[7],2DELC/MS/MSLC/MS/MS4,1175,4641952,.2nanoLC-MS/MS1011,:nanoLC/MS/MS1nanoLC/MS/MSSwiss-Protaccessionnumber(62)OGE/[5](69)[7](1175)O94832MyosinIdP00450CeruloplasminprecursorP00734ProthrombinprecursorP00747PlasminogenprecursorP00751ComplementfactorBprecursorP01008Antithrombin-IIIprecursorP01009Alpha-1-antitrypsinprecursorP01011Alpha-1-antichymotrypsinprecursorP01019AngiotensinogenprecursorP01023Alpha-2-MacroglobulinprecursorP01024ComplementC3precursorP01028ComplementC4precursorP01042Kininogen-1precursorP01243ChorionicsomatomammotropinhormoneprecursorP01833Polymeric-immnunoglobulinreceptorprecursorP01884Beta-2-microglobulinprecursorP02452Collagenalpha-1(I)chainprecursorP02647ApolipoproteinA-IprecursorP02652ApolipoproteinA-precursorP02679FibrinogengammachainprecursorP02748ComplementcomponentC9precursorP02749Beta-2-glycoproteinIprecursorP02750Leucine-richalpha-2-glycoproteinprecursorP02751FibronectinprecursorP02760AMBPproteinprecursorP02763Alpha-1-acidglycoprotein1precursorP02765Alpha-2-HS-glycoproteinprecursorP02766TransthyretinprecursorP02768SerumalbuminprecursorP02774VitaminD-bindingproteinprecursorP02787SerotransferrinprecursorP02790HemopexinprecursorP04004VitronectinprecursorP04080Cystatin-B(Stefin-B)P04114ApolipoproteinB-100precursorP04217Alpha-1B-glycoproteinprecursorP04264Keratin,typeIIcytoskeletal1P05154PlasmaserineproteaseinhibitorprecursorP05362Intercellularadhesionmolecule1precursorP05543Thyroxine-bindingglobulinprecursorP06702CalgranulinBP06727ApolipoproteinA-IVprecursorP07093GliaderivednexinprecursorP07911UromodulinprecursorP07988Pulmonarysurfactant-associatedproteinBprecursorP08185Corticosteroid-bindingglobulinprecursorP08697Alpha-2-antiplasminprecursorP08833Insulin-likegrowthfactorbindingprotein1precursorP09871ComplementC1ssubcomponentprecursorP10451OsteopontinprecursorP10909Clusterinprecursor201()29()Swiss-Protaccessionnumber(62)OGE/[5](69)[7](1175)P11464Pregnancy-specificbeta-1-glycoprotein1precursorP11684UteroglobinprecursorP12830Epithelial-cadherinprecursorP13535Myosinheavychain,skeletalmuscleperinatalP13987CD59glycoproteinprecursorP14543Nidogen-1precursorP15144AminopeptidaseN(EC3.4.11.2)P19652Alpha-1-acidglycoprotein2precursorP19801Amiloride-sensitiveamineoxidase[coppercontaining]precursor(EC1.4.3.6)P19823Inter-alpha-trypsininhibitorheavychainH2precursorP20142Gastricsinprecursor(EC3.4.23.3)P24043Lamininalpha-2chainprecursorP25054Adenom