Thermo的离子阱和蛋白质组学.ppt

蛋白质组学与生物质谱技术,主要内容,蛋白质组学概念及主要研究内容蛋白质组学技术基础蛋白质组学面临的挑战及其对策蛋白质鉴定的主要策略及技术流程样品前处理色谱/电泳分离质谱分析数据库检索,ProteomeandProteomics,一个基因组、一个细胞或组织所表达的全部蛋白质(PROTEOME=PROTEin+genOME)Proteomics通过研究蛋白质组揭示基因组序列和细胞行为之间的关系蛋白质组学是发现的科学，是系统生物学的重要组成部分蛋白质的鉴定是蛋白质组学研究的基本问题,Proteomicsn:ProvenThermoSolutions,ProteinIDQuantitationThroughput,MSnPhosphorylationGlycosylation,IntactProteinAnalysisProteinIsoformsBiomarkers,DifferentialExpressionLabelsLabel-free,Bottum-upandTop-down,在蛋白质的水平上进行的蛋白质组学研究理想的蛋白质组学研究模式,当前技术攻关的热点技术上存在困难,近期难以实现通量化在多肽的水平上进行的蛋白质组学研究技术相对成熟,是当前蛋白质组学研究的主流信息不全,蛋白质组学的基础和前提,双向电泳和多维液相色谱技术生物质谱技术基因组计划的完成生物信息学技术,Promiseofproteomics,Rodriguez-Ortegaetal.NatBiotechnol.2006Feb;24(2):191-7.,蛋白质组的复杂性vs蛋白质组学中的分离策略,2DE在蛋白水平上的分离2DLC在多肽水平上的分离,Challengesinproteomics,Lookdeeper,1mL,1L,1L,1,000L或1L起始体积MS，1amolLOD,血浆样品起始体积MS，1fmolLOD,Albumin,IgG,tissueleakingproteins,Interleukinscytokines,Fibronectin,ComplementC1,10-3/mili,10-6/micro,mol/L,10-9/nano,10-12/pico,10-15/femto,10-18/atto,10-21/zepto,10-24/yocto,InsulinPeptidedrugs,5106,5103,ng/mL,averageM.W.=5kDa,5100,510-3,510-6,510-9,unknowns,Proteindrugs,质谱,Science.2006,Vol312,212-217,离子源,EICIFABAPCIESIMALDIAPPI,IonTransferTube,ESINeedle,+/-5kV,TaylorCone,SolventevaporationandIondesolvation,ESI:Electrosprayionization,MALDI：matrixassistedlaserdesorptionionization,质量分析器,Singlequadrupole单四极杆Triplequadrupole三级四极杆Iontrap离子阱TOF飞行时间OrbitrapFTICRQ-TOFTOF-TOFQ-trapIontrap-TOFLTQ-OrbitrapLTQ-FT,裂解方式,CID/CAD碰撞诱导裂解PSD源后裂解SID/sourcefragmentation源内裂解IRMPDECDETDPQD,Whytrypsin?,Trypsin的作用位点在K、R的C-端肽键，专一性很强，水解蛋白产生的多肽（trypticpeptide）多在2-3kDa，每个蛋白平均约产生50个肽段左右。TrypticPeptide在ESIpositivemode下离子化效率高质谱在此质量范围能够准确测量，加上较多的肽段数及很强的专一性，保证了PMF策略鉴定蛋白质的可行性有效的MS/MS图谱一般来自大小在2-3kDa的多肽，且以R、K结尾的多肽裂解充分，有较强的规律性，保证了MS/MS策略的可行性。,PMFvs.MS/MSsearching,PMF可以提供较高的序列覆盖率多采用2DE-MALDI-MS配置，适用于简单样品没有序列信息，可靠性差已经过时MS/MS提供序列信息，可靠性高多采用LC-ESI-MS/MS配置，适用于复杂样品存在采集时间限制问题广为接受,SeparationpowerPeakcapacities,*measurementsperhour,2DE500-10,000proteinspotsAffinityChromatography2IonexchangeChromatography10proteinlevelGelfiltration50mg工业规模2.5lofplasma(K.Roseetal.inProteomics704),但是，也不要上得太多!,TandemMS,DatadependentMS/MS,基本原则先做一次全扫描（fullMS)，记录个丰度最高的离子然后依次对这X个离子进行CID,做MS/MS(fullMS2)然后再做全扫描,下一个datadependentMS/MS开始Dynamicexclusion某个离子在做了一定次数（比如2次）的MS/MS后，将在一定时间内（比如3min）不再作为侯选离子。,自动LC/MS/MS肽序列测定,+,多肽的裂解,碎片离子的命名,Residuename(A-Z)monoisotopicmassaveragemassA71.0371171.0788B114.53493114.59625C103.00919103.1388D115.02694115.0886E129.04259129.1155F147.06841147.1766G57.0214657.0520H137.05891137.1412I113.08406113.1595J0.00.0K128.09496128.1742L113.08406113.1595M131.04049131.1925N114.04293114.1039O0.00.0P97.0527697.1167Q128.05858128.1308R156.10111156.1876S87.0320387.0782T101.04768101.1051U150.95364150.034V99.0684199.1326W186.07931186.2133X111.0111.0Y163.06333163.1760Z128.55059128.62315,氨基酸残基质量,CID条件下trypticpeptides裂解规律mobileprotonmodel,在低能CID条件下，trypticpeptides主要产生以y离子和b离子为主的碎片离子此外还经常可以看到y离子、b离子中性丢失NH3或H2O的峰脯氨酸（P）残基N-端侧的肽键特别脆弱，因此含P的多肽MS/MS图谱一般会有一个脯氨酸N-端侧的肽键断裂产生的特别强y离子。而脯氨酸（P）残基C-端侧的肽键一般不断，因此相应的碎片离子基本不出现如果肽段中R数小于其所带质子（H+），肽段将以chargedirected方式裂解，碎片离子丰富；反之，则以chargeremote方式裂解，碎片离子较少。在chargeremote方式下D、E等酸性氨基酸残基C-端肽键易断糖肽上的寡糖基、磷酸肽上的磷酸基在CID条件下更容易脱落，因此该类多肽的MS/MS图谱的basepeak一般为母离子脱去修饰基团后产生的,MS/MS图谱举例,PhosphorylatedpeptideMS/MSexample,LPSIS*DLDS*IFGPVLSPK,ProminentlossofphosphoricacidfromM+2H2+ion,ESI-MS/MSvsMALDI-MS/MSwhyLTQ/LCQtypeinstrumentsopopularinproteomics?,数据库检索鉴定蛋白的过程,首先确定母离子的质量在数据库中按一定误差检索符合条件的肽段将MS/MS中观察到的子离子质量与数据库中多肽的子离子理论值比较找到最匹配的SequestMascot,FinniganIonTrapProductPortfolioTechnologyyoucantrust,BuiltoninnovationsProvenbyourcustomers,PERFORMANCE,Exceptionalcoverage,HighsensitivityMSn,FinniganLTQFT,UltimatePerformance,RedefiningAccurateMass,FinniganLTQOrbitrap,RedefiningPerformance,Blackler,A.R.etcAnal.Chem.;2006;78(4);1337-1344.VivekaMayya,KarimRezaul,Yu-ShengCong,andDavidHan.Molecular&CellularProteomics4:214223,2005.,Moreproteinidentifications,lowfalsepositives,FinniganLTQFTUltraHighPerformance,IonTrapbasedFourierTransformICRMassSpectrometerFTICR(FTMS)atLCtimescaleAccurateMassHighSensitivityUltra-highResolutionAboveAll-SimpletoOperate,LTQOrbitrapHighPerformance,AdvancedLC-MSProteomicsMetabolomicsPharmaceuticalDrugDiscoverySmallMoleculeandStructureElucidationUltra-traceLevelAnalysis,ThermosNewETDSystem,SoftwareforProteomics,BiomarkerResearch,Robotics,Centrifuges,Concentrators,LiquidChromatography/MassSpectrometry,ProteinIdentification,ProteinDigest,SampleHandling,DataAnalysis,PeptideSeparation,DataStorage,LabInformationManagementSystem,MicroplateReaders,ProteinFractionation,IntegratedWorkflows,Freezers,QuantitativeProteomicsandBiomarkerDiscoveryandValidation,CompanyConfidential,AWordAboutProteinQuantification,Itsajungleoutthere,SILAC,AQUA,cICAT,iTRAQ,IsotopeLabels,Isotope-CodedTags,ICATandiTRAQareregisteredtrademarksofAppleraCorporationAQUAisaregisteredtrademarkofHarvardUniversity,ThermoSupportsALLQuantificationMethods,IsotopeICAT,cICAT,iTRAQTagsGlobal,Complex,Expensive,Non-parallelworkflowLimitedpracticalsuccessIsotopeSILAC(aminoacids)LabelsMetabolicLabeling-EnrichedMedia(15N)Global,InexpensiveLimitedtocellsgrownincultureMethodofChoiceforBiologicalHypothesisTestingAQUALabeledinternalstandardforeveryproteinofinterestBestsensitivity,accuracyandprecisionMethodofChoiceforBiomarkerValidationLabel-MSPeakAreaRatiosFreeGlobal,“Free”MethodofChoiceforBiomarkerDiscovery,AListofPutativeMarkers-WhatNext?,ValidationRatherthancontinuetolookforallproteins,developamethodtolookonlyatalistoftargetedproteins.Immunoassay?MultiplexedMSanalysisofLabeledPeptides?StructuralCharacterizationExactproteinisoformTopDownanalysis,The“PeptideHypothesis”,Foreveryprotein,thereareanumberofpeptideswhichcanbeusedtospecificallyidentifythatprotein.Thesearecalled“ProteotypicPeptides”,Inashotgunsequencingexperiment,basedonLC/MSdata,theseareexpectedtobe1)Trypticpeptides2)Withoutcommonlymodifiedaminoacids(M,S,T,Y)3)GoodionizationefficiencyforMS/MSanalysis,-ReudiAebersold,PROTEIN-AQUAinPractice,IdentifyProteotypicPeptidesforproteinsofinterestOrder/synthesizeisotopicallyenrichedinternalstandardsforthesepeptidesSpikeaknownamountofthelabeledpeptide(s)intosampleDigestsamplePerformLC-MS/MS,followingboththelabeledinternalstandard(s)andnon-labeledpeptide(s)ofinterest(LC-SRM)Calculatepeakarearatioforstandardandunlabeledpeptide,Performance,Massrangem/z30-3000300H-SRMtransitionsMulti-residuescreeningmethodsFAIMSenabled(foodresidues),TopDownProteinAnalysis,Intactproteinanalysisdeterminesexactproteinisoform,ExactlyWhatProteinFormisPresent?,ETD:ElectronTransferDissociationaNewFragmentationMechanismforProteomics,OrganizationofEukaryoticDNA:Histones,Nuclearmembrane,Chromatinfiber,Nuclear,pore,Nucleosom