肝脏蛋白质组计划的实施与毒理学发展的机遇 (PPT )

国际肝脏蛋白质组学研究

计划的实施与毒理学发展机遇贺福初/孙启鸿军事医学科学院复旦大学生物医学研究院2005.09.21/沈阳报告提要蛋白质组学产生的时代背景“人类蛋白质组计划”的目标与意义蛋白质组学与毒理学/环境医学曼哈顿原子弹研制计划人类基因组计划阿波罗登月计划人类历史上的三大科技工程1941.12.6—1945.7.16罗斯福批准,耗资20亿美元原子半径10-10m原子体积10-30m3人体半径100m人体体积100m3太阳系半径1012m太阳系体积1034m31990.10.1-2003.4.23克林顿、布莱尔批准，耗资30亿美元1961.5.25—1969.7.20肯尼迪批准,耗资240亿美元人类基因组计划开创了

“基因组时代”基因组学向蛋白质组学“求助”!

Nature409:747,15Feb.2001

Andnowforproteome

“现在轮到蛋白质组”Science291:1221,16Feb.2001ProteomicsinGenomeland“基因组大地中的蛋白质组学”PROTEIN转录组一种细胞、组织或生物体所对应的全套mRNARNA基因组生物体所拥有的全套染色体上的全部基因DNA蛋白质组一种细胞、组织或生物体所对应的全套蛋白质PROTEIN功能执行体遗传信息载体基因和蛋白质并不存在严格的线性关系

ORF并不预示一定存在相对应的功能性基因

mRNA水平并非与蛋白质的表达水平对应翻译后修饰及同工蛋白质(Isoforms)等现象在基因水平无从表现蛋白质与蛋白质的相互作用难以在基因水平得以认识基因组与转录组不能取代蛋白质组翻译后修饰相互作用构象变化翻译后拼接移位胞质胞核蛋白质调节的多样性生命的“万花筒”蛋白质功能的群体性绳墙扇茅蛇树蛋白质作用的整体性探索生命奥秘的直接对象蛋白质组生命体的统一性源于基因组生命体的多样性、复杂性、功能性、表型源于蛋白质组1463-4x1043x109103(1012)

Proteomicsseekstoprovidefunctionalinformationforallproteins.proteomicsismoreofaconceptthanadefinedtechnology,anditreferstoanyprotein-basedapproachthathasthecapacitytoprovidenewinformationaboutproteins

onagenomewidescale.“Proteomicsincludesnotonlytheidentificationand

quantificationofproteinsbutalsothedeterminationof

their:

localization

modifications

interactions

activities

function报告提要蛋白质组学产生的时代背景“国际人类肝脏蛋白质组计划”的目标与意义蛋白质组学与毒理学/环境医学DNA序列图≠基因图人类基因组中1/3以上基因未曾确认基因确认的基本层次--蛋白质水平天书解读天书Science291:1221,2001全面揭示重大疾病发生发展机制的基础蛋白质组单基因病—遗传病多基因病—肿瘤等

重大疾病发生发展机制单一基因单一蛋白基因群蛋白质群转录组蛋白质组????发现大量新型药靶与新药的源泉蛋白质组最重要的疾病100-150每种疾病相关的基因10每个基因相关的蛋白质3–10药靶蛋白总数

3000-15,000

现有药物约2000种85％是针对目前已知的500种药靶6-30倍药靶有待发现启动人类蛋白质组计划势在必行！探索人体生命奥秘的直接对象全面揭示重大疾病发生发展机制的基础发现大量新型药靶与新药的源泉人类基因组序列及基因注释人类蛋白质组VIP:VeryImportantProteome人类蛋白质组计划的

主要科学目标验证人类基因组计划推测的基因，注释基因组阐明蛋白质组的调控模式并与转录组进行对比建立蛋白质群/组装体，蛋白质复合物或蛋白质机器,即连锁图建立人体生理学、病理学的蛋白质组基础1463-4x1043x109103(1012)

基因组计划蛋白质组计划Bodyfluidmostaccessibleforbiomarkerdiscoveryinanimalandhumans.LiverKidneyOrganBodyFluidBrainImmuneOrgans/VesselsIntestineOtherTissuesCSFFecesEndocrine/ParacrineSecretionsBileUrineAir/BALFLymphLungsEyesSkinOralCavitySweatSalivaTearsVenousBloodArterialBloodSerum10%90%SerumProteinsNon-InformativeAbundantProteinsi.e.albumin,IgG,etc.InformativeLowAbundanceProteinsEnrichmentStrategiesforInformativeSerumProteins:SELDI

SerumImmunosubtraction

人类肝脏蛋白质组计划里程碑蛋白表达谱蛋白连锁图亚细胞定位图修饰谱人类基因组计划

里程碑遗传图物理图序列图InitiativesofHumanProteomeProject(HPP)HPPPHumanPlasmaProteomeProject,USAHLPPHumanLiverProteomeProject,ChinaPSIProteomicsStandardsInitiativeUK

HBPP

HumanBrainProteomeProject,GermanyHAIHumanAntibodyInitiativeSwedenWhyisliver?wwwww.HLPP.HUPO为何研究肝脏?肝脏功能的多样性与重要性能量——能量转换、储存物质——代谢（活性分子的合成、毒性分子的分解）信息——信息分子的合成与分泌肝脏为其它组织提供能量—可氧化底物组织的活力依赖于高能键的连续生成。肝脏产生大部分脂肪酸，后者是禁食状态下能量的基本来源。肝脏是给食状态下由过剩糖合成脂肪酸的主要部位。药物毒物营养物生物异源物生物转化化学多样性向体内的生物转化体系提出严峻挑战1.2×107种以上的化学物(CAService’slist)以每周约8000种的速率增加常用化学物在63,000种以上大约11,500种，作为食物或药物制剂添加物摄入其余的50,000种，为潜在的环境污染物人类发育过程中造血组织的兴替造血系统的发育必需肝脏的“培育”合成大多数循环血浆蛋白合成和分泌血浆蛋白是肝脏实质细胞——肝细胞的独有功能（肝细胞占肝脏总细胞数的60%及肝脏质量的约80%）最有特征的血浆蛋白是白蛋白，在大多数哺乳动物中占总血浆蛋白的55-60%凝血酶、抗凝因子、溶栓酶等肝脏再生机体再生能力最强的器官终生保持旺盛的再生能力肝脏中包含的“组”(-ome)Metabolicgenomics→Metabolome

(代谢组)Energygenomics→Energome

(能量组)Pharmacogenomics→Pharmacome

(药理组)Toxicogenomics→Toxicome

(毒理组)Regeneratinggenomics

→Regenerome

(再生组)全球及中国肝炎的流行病学统计

全球:3.5亿携带者中国:1.8亿携带者死亡:23万人/年疾病负担:500亿元人民币治疗手段:抗病毒,保肝降 酶,抗纤维化,免疫治疗等缺点:病毒易反弹,病 情易反复肝炎向肝癌的恶性转化

难以遏制全世界现有3.5亿慢性乙肝病毒（HBV）携带者，占世界人口的5%，亚洲和非洲HBV携带率为8-15%

HBV携带者中50-70%病毒复制活跃，为慢性乙肝

慢性乙肝病人肝硬化发生率为2-20%，代偿性肝硬化发展成失代偿性肝硬化为20-23%，发展成肝癌的占6-15%中国的慢性乙肝病人中，约25-40%最终将死于肝硬化或合并肝癌HBV携带者最终死于相关肝病的危险性，男性为50%，女性为15%

全球:100万人/年中国:50万人/年死亡:约45万人/年疾病负担:400亿元人民币治疗手段:手术切除(只有 15%-20%)治疗效果:术后易转移,易复 发,五年存活率 40-50%全球及中国肝癌的流行病学统计原发性肝癌的治疗水平亟待突破世界上每年有100万新发原发性肝癌病人，其中40-50%在我国

我国原发性肝癌发病率和死亡率均位居第二位

近20年来我国肝癌的发病率上升近40%原发性肝癌一般起病较隐匿，早期缺乏典型临床表现，初诊大多已属中晚期

初诊肝癌病人中，只有15-20%的病人具备手术条件这些病人术后5年生存率仅为40-50%

肝癌是致死率最高的恶性肿瘤之一重大肝病的发生发展无法归咎于少数几个基因或蛋白质所呈现的功能状态和信号通路从蛋白质组层面上“全景式”揭示肝脏疾病的生理病理机制是解决重大肝病的根本出路肝炎病毒肝脏/肝细胞肝炎肝硬化/纤维化原发肝癌肝癌转移多因素、多步骤的发病机制肝脏是人类蛋白质组计划的首批目标！Prometheusstolefirefromthegodsandgaveittomortals.PrometheusBoundHeraclesLiberatePrometheusHLPP—ModernPrometheusMythPrometheus

LiverEagleLiverdiseases

HeraclesHLPP人类肝脏蛋白质组计划

HumanLiverProteomeProject(HLPP)国际HLPP共同体Generateanintegrativeapproachleadingtoacomprehensivefunctionalmapoftheliver

Expandliverproteometoits“PHYSIOME”and“PATHOME”todramaticallyacceleratethedevelopmentofdiagnostics,preventionandtherapeuticstowardsitsdiseases

Developstandardoperatingprocedures(SOPs)forotherHUPOInitiativesVISIONHUPOWorkshop,Bethesda,April28-29,2002

BroadinterestsandsupportsforinitiatingHLPP

HUPOLiverProteomeWorkshop,Beijing,October22-24,2002

GettingconsensusviewforscientificobjectivesofHLPP

HUPO1stWorldCongress,Versailles,November21-24,2002

Co-chairsofHLPP:Drs.FuchuHe,JohnBergeronandChristianBréchot

HLPPPlanningCommittee:17members;May2003ProposalsaboutWorkingPlanofHLPP

Dr.JohnBergeron:Jan31,2003(Management);

Dr.FuchuHe:Feb17,2003(ScientificStrategy)Dr.FuchuHe:Mar22,2003(ActionPlan);

Dr.ChristianBrechot:Mar31,2003(Sampling)

HLPPOffice,March18,2003HUPOLPPWorkshop,Bethesda,July17-18,2003

NIHparticipatingHUPO2ndWorldCongress,Montreal,Oct.8-12,2003

Dr.FuchuHewasappointedastheexecutiveChairofHLPP(2003-2005)HUPOHLPPSatelliteMeeting,Montreal,Oct.12,2003SetupExecutiveCommittee&9Sub-committees,ActionPlanatPilotPhaseFrenchliversampleswerecollectedanddistributedamong8labs,May,2004HUPOHLPPSatelliteMeeting,Beijing,Oct.24,2004ChinesepartofHLPPwasofficiallylaunched,Nov.,2004Chineseliversampleswerecollectedanddistributedamong7labs,Feb.,2005MISION表达谱修饰谱定位图连锁图样品库抗体库数据库HLPP的科学目标---肝脏蛋白质组的“太阳系”3-D图ORF组肝脏蛋白质组与

肝脏生理、病理的衔接肝脏蛋白质组代谢组毒理组药理组再生组肝炎组肝癌组能量组作为分泌器官，肝脏合成大多数的循环血浆蛋白

肝脏转录组“肝脏蛋白质组计划”和“血浆蛋白质组计划”的整合肝脏蛋白质组血浆蛋白质组?基因组肝脏蛋白质组肝脏转录组基因组、转录组和蛋白质组的集成证实推测基因对比调控模式

重要功能蛋白质肝病药物靶标肝病诊断标志物人类肝脏蛋白质组肝炎病毒肝脏/肝细胞感染肝炎肝硬化/纤维化原发肝癌肝癌转移重大肝病预警、诊断、预防、治疗的关键环节“东亚病夫”“肝病大国”1亿多肝炎病毒携带者1000亿多/年医疗费用SOPsSpecimenBankingPlatformsWorkingteamGlobalCollaborationExpressionProfileORFeome

AntibodiesBioinformatics…

ModificationProfileSubcellularLocalizationProtein-ProteinInteractionAntibodiesBioinformaticsIntegrateproteomewithtranscriptomeCompareproteomeswithinhealthanddiseasedliverTimeLinesPilotPhase(2003-2005)PhaseII(2006-2010)OutlineBackgroundProgressoftheHLPP-ProgressreportonexpressionprofilingInitiationoftheCNHLPPNextworkProgress-ExpressionprofilingEvaluationoftechnologiesBioinformaticsset-upSamplingandpreparationPreviewwithhumanfetalliverPrimaryanalysisofFrenchlivertissuesProgressEvaluationoftechnologiesBioinformaticsset-upSamplingandPreparationPreviewwithhumanfetalliverPrimaryanalysisofFrenchlivertissuesMajorconclusionsfromthevaluationoftechnologies–16standardproteins/7labsHighmolecularweightandlowabundanceproteinsarelessdetectableby2DE.Proteinswithonlythreeorderofmagnitudecouldbeidentifiedin2DE,butproteinswithfourorderofmagnitudecouldbeidentifiedinshotguntechnology.Morepeptides(redundant)wereidentifiedforhighMWproteinsinshotguntechnology.Somesimilarproteinswereidentified(notproteingroup),suggestingthecriteriafordistinguishingsimilarproteinsshouldbemorestrict.ProgressEvaluationoftechnologiesBioinformaticsset-upSamplingandPreparationPreviewwithhumanfetalliverPrimaryanalysisofFrenchlivertissuesHLPPExperimentalDatabaseMWSDataClient…GUI，WebServices,APIFilesInterchange,DMBSDumpDatasynchronizeDatasubmissionExperimentreports,data,files…

HLPPDataMirrorMWSDataClientMWSDataClientMWSDataClientMWSDataClientMWSServerAdministratorHLPPDataMirrorSubmissionPackagesRepositoryHLPPProjectManagementDatabaseHLPPProjectManagementSystemHLPPParticipatingLaboratoriesHLPPDataCenterTheHLPPDataManagementSystem

ArchitectureBasedonMyWorkSpaceSystem(MWS)CurrentStandarddraftofHLPPSampleInformationProteinextractionConcentrationMeasurementSeparationGel-based2DEGel1D-IEFGel1D-SDSLC-basedRPLCSCXLCSAXLCSECTrapDesaltingDigestionIonsourceMALDIAutoflexUltraflexABI4700ESIQstarQTofMicroLCQLTQMassspectrometryTOFTOFTOFUltraflexABI4700QTOFQstarQTofMicroITMSLCQLTQPeaklistgenerationABI4700QStarAutoflexTurboSequestMasslynxFlexAnalysisPeaklistpreprocessingGPSpeaklistpreprocessProtein/peptideidentificationMascotSequestProteinlistgenerationAutoflexGPSBiotoolsQstarBuildSummaryDTASelectBioworksAutoQuestProteinidentificationisbasedontheanalysisofpeptidesgeneratedbyproteolyicdigestion.Whenthedetectedpeptidesmatchmanyproteinsandcannotidentifyauniqueprotein,theresultwillbepresentedintheformofagroupofpossibleproteins.

DefinitionofProteinGROUP

CriteriaforproteinidentificationIontrapXCorr(highest)≥1.9(1),2.2(2),3.75(3);DeltaCn≥0.1;RSp≤4.ABI-TOF/TOFProteinscore≥59(Rank1forthespot)Q-TOForQ-STARProteinscore>thresholdPeptidescore>“thescoreforidentity”or29MALDI-TOFProteinscore>thresholdTheproteinsinfirstreport(multi-proteinforthemixture)ProgressEvaluationoftechnologiesBioinformaticsset-upSamplingandpreparationPreviewwithhumanfetalliverPrimaryanalysisofFrenchlivertissuesEvaluationoftheSOPsforthesubcellularfractionationObject:FindtheoptimummethodofpretreatmentSample:LivertissuesofC57miceSubcellular:Mitochondria,Golgi,PM,Nucleus,ER,CytoplasmSamplepretreatmentFreshtissuesFrozenhomogenisedtissuesFrozentissuesPurity(Westernblotting):Freshtissues>frozenhomogenizedtissues>frozentissuesTheyieldofproteins:Freshtissues>frozenhomogenizedtissues>frozentissuesIntegrity(TEM):Freshtissues>frozenhomogenizedtissuesSummaryProgressEvaluationoftechnologiesBioinformaticsset-upSamplingandPreparationPreviewwithhumanfetalliverPrimaryanalysisofFrenchlivertissuesItisagiantambitiousobjectiveandgivesagreatchallengetoembryonicproteomicsItisnecessarytocarryoutapreviewbeforetheformallaunchingofthisprojectScience302:1316,Nov.21,2003Nature425:441,Oct.2,2003AverageThroughputofthePlatform2DESystem

12Gel/3DaysAuto-SpotDigestSystem

1152spots/DayMSandMS/MSSystem

500-1000Spots/DayTheflowchartofCCPITfortheproteinexpressionprofileofhumanfetalliverTheareascalesbeforeandafterisoformprocessingingroupandproteinlevelsrespectively.Inproteinlevel,theredcircularityareasrepresentthenumberofconfirmedproteinsandtheyellowannulusareasrepresentthenumberofpossibleproteins.Non-isogroups19%Extensivelyconfirmed81%Groups41%Confirmed59%Non-isoforms43%Isoforms57%contribute

545

additionalproteins

and

eliminate763

groupscontribute

545

additionalproteins

and

eliminate1335

possible

proteinsIn

Protein

LevelIn

Group

Levelconfirmed:1950possible:

2593extensivelyconfirmed:2495non-isoformpossible:

1258ACBChromosomaldistributionofHFLproteome-encodinggenesFunctionalModulesinplasmamembraneProgressEvaluationoftechnologiesBioinformaticsset-upSamplingandPreparationPreviewwithhumanfetalliverPrimaryanalysisofFrenchlivertissues

ReferenceLabsforHLPPExpressionProfilingProject

FuchuHe&XiaohongQian(BeijingInstituteofRadiationMedicine)RongZeng(ShanghaiInstituteforBiologicalSciences)PengyuanYang(FudanUniversity)SiqiLiu(BeijingGenomicsInstitute,ChineseAcademyofSciences)LauraBeretta(FHCRC,USA)RichardJ.Simpson(RoyalMelbourneHospital,Australia)

AngelikaGorg(TechnischeUniversitatMunchen,Germany)MarkBaker/JunhongSun(APAF,Australia)OutlineBackgroundProgressoftheHLPPInitiationoftheCNHLPPNextworkCNHLPPOverviewThefirstphase:2004-2005Fundingfromcentralgovernment:10millionUSdollars ChineseMOST:jointlyfundedbyNationalProgramon KeyBasicResearchProjects(973),NationalHigh-tech R&DProgram(863),andNationalKeyTechnologiesR&D Programmeininthefirstphase.LocalandInstitutionalfunding:3.5millionUSdollars8subprojects70institutes,universities,andcompaniesinvolvedLong-termsupportasanationalproject(2006~2020)CelebrationofCNHLPPOfficialLaunching(BeijingInstituteofRadiationMedicine)KeylabsBeijingInstituteofRadiationMedicineConsultingcommittee(5)ORFeome&PPISubproject,ZeguangHanChairFuchuHeHeadquartersBPRCModificationProfilesubproject,YunCaiExpressionProfileSubproject,XiaohongQianStructuralBiologySubproject,WeiminGongBioinformaticssubproject,YixueLiNewTechnologysubproject,YukuiZhangAntibodysubproject,Qi-HongSunLocalizationMappingSubproject,XueminZhangMorethan10workshopshavebeenheldBriefprogressofsomesubprojects

ProteinmodificationProteinlocalizationProteinstructureNewproteomicstechnologiesAnalysisofproteinphosphorylationbycombinationofIMAC,PhosphatasewithBiologicalMassSpectrometrym/zPeptidemixtureProteinsCandidatephosphopeptidesIdentificationofphosphorylatedsitesDIGESTIMACMALDI-TOFMSAnalysisPhosphatasedigestMS/MSAnalysisandDatabaseSearchingm/zPhosphopeptideConfirmationm/zMetastableionsofphosphopeptidesO60716GSLApSLDpSLRKP15924GLPSPYNMSpSAPGpSRO75379NLLEDDpSDEEEDFFLRRApSpSKGGGGYTC*QSGSGWDEFTKO95210HSpSWGDVGVGGSLKP16157RDpSRDVDEEKELLDFVPKP02671ADpSGEGDFLAEGGGVRP35221TPEELDDpSDFETEDFDVRP02730

YQpSSPAKPDSSFYKQ13813WRpSLQQLAEERYQSSPAKPDSSFpYKQ15019IYHLPDAEpSDEDEDFKEQTRYQSSPAKPDSpSFYKQ16828SNIpSPNFNFMGQLLDFERRYQSSPAKPDpSSFYKQ99959RLEISPDpSpSPERAHYTHSDYQYSQRP04792GPpSWDPFRLPEEWSQWLGGSSWPGYVRPLPPAAIEpSPAVAAPAYSRQ9H3Z4SLpSTSGESLYHVLGLDKTr:O95810SLEETLHTVDLpSpSDDDLPHDEEALEDpSAEEKVEESRP05386KEEpSEEpSDDDM*GFGLFDKEEpSEEpSDDDMGFGLFDTr:Q8NEN5pSQpSLPTTLLSPVRP08559YHGHpSMSDPGVSYRTr:Q9HAP4ESEALPEKEGEELGEGERPEEDAAALELpSpSDEAVEVEEVIEESRP11277TpSPVSLWSRLpSpSSWESLQPEPSHPYTr:Q9NQA7WLDEpSDAEMELRIdentifiedphosphopeptidesequencesfromHFLProteinisolationandpurificationbylectin(Glycosylation)Co-localizationandlocomotionoftheGFPfusionproteinandDsRedDsRed-MemGreenmergeGFPGFP-fusedPrTNF-

treatedGFP-fusedmutantTNF-

treatedGFP-fusedPrStructuralproteomicsCloned900genesfromliverExpressedproductsofmorethan100genesDeterminedthestructuresof3proteinsbyX-rayDeterminedthestructuresof2proteinsbyNMRDiphosphoglyceratemutase(DPGM)X-rayProgrammedcelldeath5(PDCD5)NMRBeijing(National)ProteomeResearchCenterChineseProteomeDevelopmentCenterTheAntibodyBankofHumanLiverProteomeProjectEstablishment,characterization,andapplicationofantibodiesagainsthumanliverproteins

Qi-HongSunBeijingInstituteofRadiationMedicine(BIRM)BeijingProteomeResearchCenter(BPRC)AntigensMyelomacellsHybridomacellsmAbsDiagnosticsTherapeuticsProteomicsResearch

ProfilingIdentificationQuantificationLocalizationModificationInteractionFunctionLargeantibodycollectionforproteomicsAntibodyDatabaseAntibodyBankSamples5000liverproteinsHLPPAntibodyBankAntibodychips10%ofHLPPAntibodyBankStandardsanddatabaseofHUPOHLPP/HAIMicroarrayandotherantibody-basedtechnology90%10%HLPPAntibodyBankatPilotPhaseAntigenpreparation

Fractionatedliverproteins-Unknown/native/multi-antigensRecombinantproteinsSynthesizedpeptidesAntibodygenerationHybridomacelllines–mAbs(murine/rabbit)Polyclonalantibodies–IgG(Rabbit)/orIgY(chicken)AntibodycharacterizationandapplicationClas