肝脏蛋白质组计划的实施与毒理学发展的机遇ppt课件

肝脏蛋白质组计划的实施与毒理学发展的机遇肝脏蛋白质组计划的实施与毒理学发展的机遇1（优选）肝脏蛋白质组计划的实（优选）肝脏蛋白质组计划的实施与毒理学发展的机遇施与毒理学发展的机遇（优选）肝脏蛋白质组计划的实施与毒理学发展的机遇2曼哈顿原子弹曼哈顿原子弹研制计划研制计划人类基因组计划人类基因组计划阿波罗登月计划阿波罗登月计划人类历史上的三大科技工程人类历史上的三大科技工程1941.12.61945.7.16罗斯福批准罗斯福批准,耗资耗资20亿美元亿美元原子半径原子半径10-10m原子体积原子体积10-30m3人体半径人体半径100m人体体积人体体积100m3太阳系半径太阳系半径1012m太阳系体积太阳系体积1034m31990.10.1-2003.4.23克林顿、布莱尔批准，克林顿、布莱尔批准，耗资耗资30亿美元亿美元1961.5.251969.7.20肯尼迪批准肯尼迪批准,耗资耗资240亿美元亿美元曼哈顿原子弹人类基因组计划阿波罗登月计划人类历史上的三大科技3人类基因组计划开创了人类基因组计划开创了“基因组时代基因组时代”人类基因组计划开创了4基因组学向基因组学向蛋白质组学蛋白质组学“求助求助”!Nature409:747,15Feb.2001Andnowforproteome“现在轮到蛋白质组现在轮到蛋白质组”Science291:1221,16Feb.2001ProteomicsinGenomeland“基因组大地中的蛋白质组学基因组大地中的蛋白质组学”基因组学向Nature 409:747,15 Feb.25PROTEINPROTEIN6转录组转录组一种细胞、组织或生物体所对应的全套全套mRNA RNA RNA基因组基因组生物体所拥有的全套染色体上的全部基因DNADNA蛋白质组蛋白质组一种细胞、组织或生物体所对应的全套蛋白质PROTEINPROTEIN功能执行体功能执行体遗传信息载体遗传信息载体转录组一种细胞、组织或生物体所对应的全套mRNA RNA基因7基因和蛋白质并不存在严格的线性关系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翻译后修饰及同工蛋白质(Isoforms)等现象在基等现象在基因水平无从表现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蛋白质与蛋白质的相互作用难以在基因水平得以认识认识基因组与转录组不能取代蛋白质组基因组与转录组不能取代蛋白质组 基因和蛋白质并不存在严格的线性关系基因组与转录组不能取代蛋8翻译后修饰翻译后修饰相互作用相互作用构象变化构象变化翻译后拼接翻译后拼接移位移位胞质胞核蛋白蛋白质调节的多的多样性性生命的生命的“万花筒万花筒”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9蛋白质功能的群体性蛋白质功能的群体性蛋白质功能的群体性10Antibody databaseMore peptides(redundant)were identified for high MW proteins in shotgun technology.Nature 409:747,15 Feb.8 subprojectslocalization1152 spots/DayProtein modificationEvaluation of technologiesPrimary analysis of French liver tissuesGerman Systems Biology Project(Drs.Co-localization and locomotion of theQuantificationSiqi Liu(Beijing Genomics Institute,Chinese Academy of Sciences)IYHLPDAEpSDEDEDFKEQTRYQpSSPAKPDSSFYKAntibody-based technology绳绳墙墙扇扇茅茅蛇蛇树树蛋白质作用的整体性蛋白质作用的整体性Antibody database绳墙扇茅蛇树蛋白质作用的整11探索生命奥秘的直接探索生命奥秘的直接对象象蛋白蛋白质组 生命体的生命体的统一性源于基因一性源于基因组 生命体的多生命体的多样性、复性、复杂性、性、功能性、表型源于蛋白功能性、表型源于蛋白质组1463-4x1043x109103(1012)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112n Proteomics seeks to provide functional information for all proteins.n proteomics is more of a concept than a defined technology,and it refers to any protein-based approach that has the capacity to provide new information about proteins on a genomewide scale.n“Proteomics includes not only the identification and quantification of proteins but also the determination of their:n localizationn modificationsn interactionsn activitiesn function Proteomics seeks to provide f13报报告告提提要要蛋白质组学产生的时代背景蛋白质组学产生的时代背景“国际人类肝脏蛋白质组计划国际人类肝脏蛋白质组计划”的目标与意的目标与意义义蛋白质组学与毒理学蛋白质组学与毒理学/环境医学环境医学报 告 提 要蛋白质组学产生的时代背景14DNA序列图序列图基因图基因图人类基因组中人类基因组中1/3以上以上基因未曾确认基因未曾确认基因确认的基本层次基因确认的基本层次-蛋白质水平蛋白质水平天书天书解读天书解读天书Science291:1221,2001DNA序列图基因图天书解读天书Science 291:1215全面揭示重大疾病全面揭示重大疾病发生生发展机制的基展机制的基础蛋白蛋白质组单基因病单基因病遗传病遗传病多基因病多基因病肿瘤等肿瘤等 重大疾病发生发展机制重大疾病发生发展机制单一基因单一基因单一蛋白单一蛋白基因群基因群蛋白质群蛋白质群转录组转录组蛋白质组蛋白质组?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16Rabbit pAbs 50Enrichment Strategies for Informative Serum Proteins:CoordinationIdentificationHuman BrainFresh tissues frozen homogenized tissues frozen tissuesRDpSRDVDEEKELLDFVPK重大肝病的发生发展无法归咎于少数几个基因或Fresh tissues frozen homogenized tissues frozen tissuesHLPP ExperimentalChromosomal distribution of HFL proteome-encoding genesHLPPModern Prometheus MythCNHLPP OverviewAntibody demands/applications重大肝病的发生发展无法归咎于少数几个基因或LPEEWSQWLGGSSWPGYVRPLPPAAIEpSPAVAAPAYSRCandidate phosphopeptidesAntibody microarray and other antibody-based technologyESEALPEKEGEELGEGERPEEDAAALELpSpSDEAVEVEEVIEESR发现大量新型大量新型药靶与新靶与新药的源泉的源泉蛋白蛋白质组最重要的疾病最重要的疾病100-150每种疾病相关的基因每种疾病相关的基因10每个基因相关的蛋白质每个基因相关的蛋白质310药靶蛋白总数药靶蛋白总数3000-15,000现有药物约现有药物约2000种种85是针对目前已知的是针对目前已知的500种药靶种药靶6-30倍药靶有待发现倍药靶有待发现Rabbit pAbs 50发现大量新型药靶与新药的源泉17启启动人人类蛋白蛋白质组计划划势在必行！在必行！探索人体生命奥秘的直接探索人体生命奥秘的直接对象象全面揭示重大疾病全面揭示重大疾病发生生发展机制的基展机制的基础发现大量新型大量新型药靶与新靶与新药的源泉的源泉人人类基因基因组序列及基因注序列及基因注释人类蛋白质组人类蛋白质组VIP:VeryImportantProteome启动人类蛋白质组计划探索人体生命奥秘的直接对象全面揭示重大疾18人类蛋白质组计划的人类蛋白质组计划的主要科学目标主要科学目标验证人人类基因基因组计划推划推测的基因，注的基因，注释基因基因组阐明蛋白明蛋白质组的的调控模式并与控模式并与转录组进行行对比比建立蛋白建立蛋白质群群/组装体，蛋白装体，蛋白质复合物或蛋白复合物或蛋白质机器机器,即即连锁图建立人体生理学、病理学的蛋白建立人体生理学、病理学的蛋白质组基基础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191463-4x1043x109103(1012)基因组计划基因组计划蛋白质组计划蛋白质组计划1463-4 x 1043 x 109103(1012)20肝脏蛋白质组计划的实施与毒理学发展的机遇ppt课件21Bodyfluidmostaccessibleforbiomarkerdiscoveryinanimalandhumans.LiverKidneyOrganBody FluidBrainImmune Organs/VesselsIntestineOther TissuesCSFFecesEndocrine/Paracrine SecretionsBileUrineAir/BALFLymphLungsEyesSkinOral CavitySweatSalivaTearsVenous BloodArterial BloodSerum10%90%Serum ProteinsNon-Informative Abundant Proteins i.e.albumin,IgG,etc.Informative Low Abundance ProteinsEnrichment Strategies for Informative Serum Proteins:SELDI Serum Immunosubtraction Body fluid most accessible for22人类肝脏蛋白质组人类肝脏蛋白质组计划里程碑计划里程碑蛋白表达蛋白表达谱蛋白蛋白连锁图亚细胞定位胞定位图修修饰谱人类基因组计划人类基因组计划里程碑里程碑遗传图物理物理图序列序列图人类肝脏蛋白质组蛋白表达谱人类基因组计划里程碑遗传图23Initiatives of Human Proteome Project(HPP)HPPPHuman Plasma ProteomeProject,USAHLPPHuman Liver Proteome Project,ChinaPSIProteomics Standards Initiative UK HBPP Human Brain Proteome Project,GermanyHAIHumanAntibodyInitiativeSwedenInitiatives of Human Proteome 24Antibody databaseOral Cavity信息信息分子的合成与分泌Yukui ZhangSLpSTSGESLYHVLGLDKRegistrationAntibody demands/applications发现大量新型药靶与新药的源泉pSQpSLPTTLLSPVRSample:Liver tissues of C57 mice6-30倍药靶有待发现More peptides(redundant)were identified for high MW proteins in shotgun technology.Antibody datasheet Electronic submission formPreview with human fetal liverNature 409:747,15 Feb.Proposals about Working Plan of HLPPGeneral ApproachSynthesized peptidesPreview with human fetal liverEvaluation of technologiesMRPP,HPPP,PSI,HAI,etc.Whyisliver?wwwww.HLPP.HUPO为何研究肝脏为何研究肝脏?Antibody databaseWhy is liver?25肝脏功能的多样性与重要性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功能的多样性与重要性能量能量转换、储存26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。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27药药物物毒毒物物营养物营养物生物异源物生物异源物生物转化生物转化药 物毒 物营养物生物异源物生物转化28化学多样性向体内的生物转化体系提出严峻挑战1.2107种以上的化学物种以上的化学物(CAServiceslist)以每周以每周约8000种的速率增加种的速率增加常用化学物在常用化学物在63,000种以上种以上大大约11,500种，作种，作为食物或食物或药物制物制剂添加物添加物摄入入其余的其余的50,000种，种，为潜在的潜在的环境境污染物染物化学多样性向体内的生物转化体系提出严峻挑战1.2107种以29人类发育过程中造血组织的兴替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造血系统的发育必需肝脏的“培育培育”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”30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31肝脏再生机体再生能力机体再生能力最强的器官最强的器官终生保持旺盛终生保持旺盛的再生能力的再生能力肝脏再生机体再生能力32肝脏中包含的肝脏中包含的“组组”(-ome)Metabolic genomics Metabolome(代谢组代谢组)Energy genomics Energome(能量组能量组)Pharmacogenomics Pharmacome(药理组药理组)Toxicogenomics Toxicome(毒理组毒理组)Regenerating genomicsRegenerome(再生组再生组)肝脏中包含的“组”(-ome)Metabolic genom33全球及中国肝炎的流行病学统计全球全球:3.5亿携携带者者中国中国:1.8亿携携带者者死亡死亡:23万人万人/年年疾病疾病负担担:500亿元人民元人民币治治疗手段手段:抗病毒抗病毒,保肝降保肝降 酶,抗抗纤维化化,免疫治免疫治疗等等缺点缺点:病毒易反病毒易反弹,病病 情易反复情易反复全球及中国肝炎的流行病学统计 34肝炎向肝癌的恶性转化肝炎向肝癌的恶性转化难以遏制难以遏制全世界现有全世界现有3.53.5亿慢性乙肝病毒（亿慢性乙肝病毒（HBVHBV）携带者，占世界人口的）携带者，占世界人口的5%5%，亚洲和非，亚洲和非洲洲HBVHBV携带率为携带率为8-15%8-15%HBVHBV携带者中携带者中50-70%50-70%病毒复制活跃，为慢性乙肝病毒复制活跃，为慢性乙肝慢性乙肝病人肝硬化发生率为慢性乙肝病人肝硬化发生率为2-20%2-20%，代代偿性肝硬化性肝硬化发展成失代展成失代偿性肝硬化性肝硬化为20-23%20-23%，发展成肝癌的占展成肝癌的占6-15%6-15%中国的慢性乙肝病人中，中国的慢性乙肝病人中，约25-40%25-40%最最终将死于肝硬化或合并肝癌将死于肝硬化或合并肝癌 HBVHBV携携带者最者最终死于相关肝病的危死于相关肝病的危险性，男性性，男性为50%50%，女性，女性为15%15%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35全球全球:100万人万人/年年中国中国:50万人万人/年年死亡死亡:约45万人万人/年年疾病疾病负担担:400亿元人民元人民币治治疗手段手段:手手术切除切除(只有只有15%-20%)治治疗效果效果:术后易后易转移移,易复易复发,五年存活率五年存活率40-50%全球及中国肝癌的流行病学全球及中国肝癌的流行病学统计全球及中国肝癌的流行病学统计36原原发性肝癌的治性肝癌的治疗水平水平亟待突破亟待突破世界上每年有世界上每年有100100万新发原发性肝癌病人，其中万新发原发性肝癌病人，其中40-50%40-50%在我国在我国我国原发性肝癌发病率和死亡率均位居第二位我国原发性肝癌发病率和死亡率均位居第二位近近2020年来我国肝癌的发病率上升近年来我国肝癌的发病率上升近40%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已属中晚期初诊肝癌病人中，只有初诊肝癌病人中，只有15-20%15-20%的病人具备手术条件的病人具备手术条件这些病人些病人术后后5 5年生存率年生存率仅为40-50%40-50%肝癌是致死率最高的恶性肿瘤之一肝癌是致死率最高的恶性肿瘤之一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37重大肝病的发生发展无法归咎于少数几个基因或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蛋白质所呈现的功能状态和信号通路从蛋白质组层面上从蛋白质组层面上“全景式全景式”揭示揭示肝脏疾病的生理肝脏疾病的生理病理机制病理机制是解决重大肝病的根本出路是解决重大肝病的根本出路肝炎病毒肝炎病毒肝脏肝脏/肝细胞肝细胞肝炎肝炎肝硬化肝硬化/纤维化纤维化原发肝癌原发肝癌肝癌转移肝癌转移多因素、多因素、多步骤的发病机制多步骤的发病机制重大肝病的发生发展无法归咎于少数几个基因或从蛋白质组层面上“38肝脏是人类蛋白质组计划的首批目标！肝脏是人类蛋白质组计划的首批目标！39Prometheus stole fire from the gods and gave it to mortals.Prometheus BoundHeraclesLiberatePrometheusHLPPModern Prometheus MythPrometheus Liver Eagle Liver diseases Heracles HLPPPrometheus stole fire from the40人类肝脏蛋白质组计划Human Liver Proteome Project(HLPP)国国际HLPP共同体共同体人类肝脏蛋白质组计划Human Liver Proteom41Generate an integrative approach leading to a comprehensive functional map of the liver Expand liver proteome to its“PHYSIOME”and“PATHOME”to dramatically accelerate the development of diagnostics,prevention and therapeutics towards its diseases Develop standard operating procedures(SOPs)for other HUPO InitiativesVISIONGenerate an integrative approa42HUPOWorkshop,Bethesda,April28-29,2002BroadinterestsandsupportsforinitiatingHLPPHUPOLiverProteomeWorkshop,Beijing,October22-24,2002GettingconsensusviewforscientificobjectivesofHLPPHUPO1stWorldCongress,Versailles,November21-24,2002Co-chairsofHLPP:Drs.FuchuHe,JohnBergeronandChristianBrchotHLPPPlanningCommittee:17members;May2003ProposalsaboutWorkingPlanofHLPPDr.JohnBergeron:Jan31,2003(Management);Dr.FuchuHe:Feb17,2003(ScientificStrategy)Dr.FuchuHe:Mar22,2003(ActionPlan);Dr.ChristianBrechot:Mar31,2003(Sampling)HLPPOffice,March18,2003HUPOLPPWorkshop,Bethesda,July17-18,2003NIHparticipatingHUPO2ndWorldCongress,Montreal,Oct.8-12,2003Dr.FuchuHewasappointedastheexecutiveChairofHLPP(2003-2005)HUPOHLPPSatelliteMeeting,Montreal,Oct.12,2003SetupExecutiveCommittee&9Sub-committees,ActionPlanatPilotPhaseFrenchliversampleswerecollectedanddistributedamong8labs,May,2004HUPOHLPPSatelliteMeeting,Beijing,Oct.24,2004ChinesepartofHLPPwasofficiallylaunched,Nov.,2004Chineseliversampleswerecollectedanddistributedamong7labs,Feb.,2005MISIONHUPO Workshop,Bethesda,April43表达谱表达谱修饰谱修饰谱定位图定位图连锁图连锁图样品库样品库抗体库抗体库数据库数据库HLPP的科学目标的科学目标-肝脏蛋白质组的肝脏蛋白质组的“太阳系太阳系”3-D图图ORF组组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44SLpSTSGESLYHVLGLDK)or other programme(e.Funding from central government:10 million US dollarsRLEISPDpSpSPERAHYTHSDYQYSQR“肝脏蛋白质组计划”和The metabolism pathwaysCo-localization and locomotion of theNature 409:747,15 Feb.肝脏蛋白质组与肝脏生理、病理的衔接this projectSampling and preparationSampling and Preparationto carry out a preview beforePeptide score “the score for identity”or 29Nature 409:747,15 Feb.Criteria for protein identificationHuman Plasma ProteomeProtein extraction物质代谢（活性分子的合成、CNHLPP Overview肝脏蛋白质组与肝脏蛋白质组与肝脏生理、病理的衔接肝脏生理、病理的衔接肝脏蛋白质组肝脏蛋白质组代谢组代谢组毒理组毒理组药理组药理组再生组再生组肝炎组肝炎组 肝癌组肝癌组能量组能量组SLpSTSGESLYHVLGLDK肝脏蛋白质组与肝脏生理45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“肝脏蛋白质组计划肝脏蛋白质组计划”和和“血浆蛋白质组计划血浆蛋白质组计划”的整合的整合肝脏蛋白质组肝脏蛋白质组血浆蛋白质组血浆蛋白质组?作为分泌器官，肝脏合成大多数的“肝脏蛋白质组计划”和肝脏蛋白46基因组基因组肝脏肝脏蛋白质组蛋白质组肝脏肝脏转录组转录组基因组、转录组和蛋白质组的集成基因组、转录组和蛋白质组的集成证实推推测基因基因对比比调控模式控模式 基因组肝脏肝脏基因组、转录组和蛋白质组的集成证实推测基因对比47重要功能蛋白质重要功能蛋白质肝病药物靶标肝病药物靶标肝病诊断标志物肝病诊断标志物人类肝脏蛋白质组人类肝脏蛋白质组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48肝炎病毒肝炎病毒肝脏肝脏/肝细胞肝细胞感感染染肝炎肝炎肝硬化肝硬化/纤维化纤维化原发肝癌原发肝癌肝癌转移肝癌转移重大肝病预警、诊断、预防、治疗的关键环节重大肝病预警、诊断、预防、治疗的关键环节“东亚病夫东亚病夫”“肝病大国肝病大国”1亿多肝炎亿多肝炎病毒携带者病毒携带者1000亿多亿多/年年医疗费用医疗费用肝炎病毒肝脏/肝细胞感肝炎肝硬化/纤维化原发肝癌肝癌转移重大49qSOPsqSpecimenBankingqPlatformsqWorkingteamqGlobalCollaborationqExpressionProfileqORFeomeqAntibodiesqBioinformaticsqqModificationProfileqSubcellularLocalizationqProtein-ProteinInteractionqAntibodiesqBioinformaticsqIntegrateproteomewithtranscriptomeqCompareproteomeswithinhealthanddiseasedliverTimeLinesPilotPhase(2003-2005)PhaseII(2006-2010)SOPs Modification ProfileTime 50OutlineBackgroundProgressoftheHLPP-ProgressreportonexpressionprofilingInitiationoftheCNHLPPNextworkOutline Background51Progress-ExpressionprofilingEvaluationoftechnologiesBioinformaticsset-upSamplingandpreparationPreviewwithhumanfetalliverPrimaryanalysisofFrenchlivertissuesProgress-Expression profilin52ProgressEvaluationoftechnologiesBioinformaticsset-upSamplingandPreparationPreviewwithhumanfetalliverPrimaryanalysisofFrenchlivertissuesProgressEvaluation of technolo53Majorconclusionsfromthevaluationoftechnologies16standardproteins/7labsnHighmolecularweightandlowabundanceproteinsarelessdetectableby2DE.nProteinswithonlythreeorderofmagnitudecouldbeidentifiedin2DE,butproteinswithfourorderofmagnitudecouldbeidentifiedinshotguntechnology.nMorepeptides(redundant)wereidentifiedforhighMWproteinsinshotguntechnology.nSomesimilarproteinswereidentified(notproteingroup),suggestingthecriteriafordistinguishingsimilarproteinsshouldbemorestrict.Major conclusions from the val54ProgressEvaluationoftechnologiesBioinformaticsset-upSamplingandPreparationPreviewwithhumanfetalliverPrimaryanalysisofFrenchlivertissuesProgressEvaluation of technolo55HLPPExperimentalDatabaseMWSDataClientGUI，WebServices,APIFilesInterchange,DMBSDumpData synchronizeData submissionExperiment reports,data,files HLPPDataMirrorMWSDataClientMWSDataClientMWSDataClientMWSDataClientMWSServerAdministratorHLPPDataMirrorSubmissionPackagesRepositoryHLPPProjectManagementDatabaseHLPPProjectManagementSystemHLPPParticipatingLaboratoriesHLPPDataCenterTheHLPPDataManagementSystemArchitectureBasedonMyWorkSpace System(MWS)HLPP Experimental MWS Data Cli56CurrentStandarddraftofHLPPSampleInformationProteinextractionConcentrationMeasurementSeparationGel-based2DEGel1D-IEFGel1D-SDSLC-basedRPLCSCXLCSAXLCSECTrapDesaltingDigestionIonsourcenMALDIAutoflexUltraflexABI4700nESIQstarQTofMicroLCQLTQMassspectrometrynTOFnTOFTOFUltraflexABI4700nQTOFQstarQTofMicronITMSLCQLTQPeaklistgenerationABI4700QStarAutoflexTurboSequestMasslynxFlexAnalysisPeaklistpreprocessingGPSpeaklistpreprocessProtein/peptideidentificationMascotSequestProteinlistgenerationnAutoflexnGPSnBiotoolsnQstarnBuildSummarynDTASelectnBioworksnAutoQuestCurrent Standard draft of HLPP57初诊肝癌病人中，只有15-20%的病人具备手术条件GFP-fused PrScience 302:1316,基因组与转录组不能取代蛋白质组WLDEpSDAEMELRSerum ImmunosubtractionThe metabolism pathwaysMyeloma cellsInitiation of the CNHLPP“国际人类肝脏蛋白质组计划”的目标与意义Yukui ZhangProteomic technologies are being used to assess global changes in protein expression in order to identify biomarkers of chemical exposure and environmentally related diseases.HLPP Planning Committee:17 members;May 2003HUPO 2nd World Congress,Montreal,Oct.蛋白质所呈现的功能状态和信号通路Protein localizationIt is a giant ambitious objective and gives a great challenge to embryonic proteomicsHuman BrainHuman Plasma ProteomeQ-TOF or Q-STARFresh tissues frozen homogenized tissues frozen tissuesProtein identification is based on the analysis of peptides generated by proteolyic digestion.When the detected peptides match many proteins and cannot identify a unique protein,the result will be presented in the form of a group of possible proteins.DefinitionofProteinGROUP初诊肝癌病人中，只有15-20%的病人具备手术条件Prote58CriteriaforproteinidentificationnIontrapXCorr(highest)1.9(1),2.2(2),3.75(3);DeltaCn0.1;RSp4.nABI-TOF/TOFProteinscore59(Rank1forthespot)nQ-TOForQ-STARProteinscorethresholdPeptidescore“thescoreforidentity”or29nMALDI-TOFProteinscorethresholdTheproteinsinfirstreport(multi-proteinforthemixture)Criteria for protein identific59ProgressEvaluationoftechnologiesBioinformaticsset-upSamplingandpreparationPreviewwithhumanfetalliverPrimaryanalysisofFrenchlivertissuesProgressEvaluation of technolo60EvaluationoftheSOPsforthesubcellularfractionationObject:FindtheoptimummethodofpretreatmentSample:LivertissuesofC57miceSubcellular:Mitochondria,Golgi,PM,Nucleus,ER,CytoplasmSamplepretreatmentFreshtissuesFrozenhomogenisedtissuesFrozentissuesEvaluation of the SOPs for the61Purity(Westernblotting):FreshtissuesfrozenhomogenizedtissuesfrozentissuesTheyieldofproteins:FreshtissuesfrozenhomogenizedtissuesfrozentissuesIntegrity(TEM):FreshtissuesfrozenhomogenizedtissuesSummaryPurity(Western blotting):Fre62ProgressEvaluationoftechnologiesBioinformaticsset-upSamplingandPreparationPreviewwithhumanfetalliverPrimaryanalysisofFrenchlivertissuesProgressEvaluation of technolo63ItisagiantambitiousobjectiveandgivesagreatchallengetoembryonicproteomicsItisnecessarytocarryoutapreviewbeforetheformallaunchingofthisprojectScience302:1316,Nov.21,2003Nature425:441,Oct.2,2003It is a giant ambitious object64AverageThroughputofthePlatform 2DE System 12Gel/3Days Auto-Spot Digest System 1152spots/Day MS and MS/MS System500-1000Spots/DayAverage Throughput of the Plat65TheflowchartofCCPITfortheproteinexpressionTheflowchartofCCPITfortheproteinexpressionprofileofhumanfetalliverprofileofhumanfetalliverThe flowchart of CCPIT for the66 The area scales before and after isoform processing in group and protein levels respectively.In protein level,the redcircularityareas represent the number of confirmed proteins and the yellowannulusareas represent the number of possible proteins.Non-isogroups19%Extensivelyconfirmed81%Groups41%Confirmed59%Non-isoforms43%Isoforms57%contribute545additionalproteinsandeliminate763groupscontribute545additionalproteinsandeliminate1335possibleproteinsInProteinLevelInGroupLevelconfirmed:1950possible:2593extensivelyconfirmed:2495non-isoformpossible:1258 The area scales before and67ACBChromosomaldistributionofHFLproteome-encodinggenesACBChromosomal distribution of68Why is liver?“国际人类肝脏蛋白质组计划”的目标与意义肝脏蛋白质组计划的实施与毒理学发展的机遇HBPPFresh tissues frozen homogenized tissues frozen tissuesProtein score thresholdHLPP Office,March 18,2003Co-localization and locomotion of thehas the capacity to provide new information about proteins重大肝病预警、诊断、预防、治疗的关键环节基因组、转录组和蛋白质组的集成Funding from central government:10 million US dollarsMWS Data Client5000 liver proteinsGenerate an integrative approach leading to a comprehensive functional map of the liver Expand liver proteome to its“PHYSIOME”and“PATHOME”to dramatically accelerate the development of diagnostics,prevention and therapeutics towards its diseases Develop standard operating procedures(SOPs)for other HUPO InitiativesCo-localization and locomotion of theEnergy genomics Energome(能量组)Evaluation of technologiesglobal protein analysis.FunctionalModulesinplasmamembraneWhy is liver?Functional Module69ProgressEvaluationoftechnologiesBioinformaticsset-upSamplingandPreparationPreviewwithhumanfetalliverPrimaryanalysisofFrenchlivertissuesProgressEvaluation of technolo70ReferenceLabsforHLPPExpressionProfilingProjectFuchuHe&XiaohongQian(BeijingInstituteofRadiationMedicine)RongZeng(ShanghaiInstituteforBiologicalSciences)PengyuanYang(FudanUniversity)SiqiLiu(BeijingGenomicsInstitute,ChineseAcademyofSciences)Reference Labs for HLPP Expre71LauraBeretta(FHCRC,USA)RichardJ.Simpson(RoyalMelbourneHospital,Australia)AngelikaGorg(TechnischeUniversitatMunchen,Germany)MarkBaker/JunhongSun(APAF,Australia)Laura Beretta Richard J.Simps72OutlineBackgroundProgressoftheHLPPInitiationoftheCNHLPPNextworkOutline Background73CNHLPPOverviewThefirstphase:2004-2005Fundingfromcentralgovernment:10millionUSdollarsChineseMOST:jointlyfundedbyNationalProgramonKeyBasicResearchProjects(973),NationalHigh-techR&DProgram(863),andNationalKeyTechnologiesR&DProgrammeininthefirstphase.LocalandInstitutionalfunding:3.5millionUSdollars8subprojects70institutes,universities,andcompaniesinvolvedLong-termsupportasanationalproject(20062020)CNHLPP OverviewThe first phase74CelebrationofCNHLPPOfficialLaunching(BeijingInstituteofRadiationMedicine)Celebration of CNHLPP Official75KeylabsBeijingInstituteofRadiationMedicine Key labsBeijing Institute of76Consultingcommittee(5)ORFeome&PPISubproject,ZeguangHanChairFuchuHeHeadquartersBPRCModificationProfilesubproject,YunCaiExpressionProfileSubproject,XiaohongQianStructuralBiologySubproject,WeiminGongBioinformaticssubproject,YixueLiNewTechnologysubproject,YukuiZhangAntibodysubproject,Qi-HongSunLocalizationMappingSubproject,XueminZhangConsulting committeeORFeome&77Morethan10workshopshavebeenheldMore than 1078BriefprogressofsomesubprojectsProteinmodificationProteinlocalizationProteinstructureNewproteomicstechnologiesBrief progress of some subproj79Analysis of protein phosphorylation by combination Analysis of protein phosphorylation by combination of IMAC,Phosphatase with Biological Mass of IMAC,Phosphatase with Biological Mass SpectrometrySpectrometrym/zPeptide mixturePeptide mixtureProteinsProteinsCandidate Candidate phosphopeptidesphosphopeptidesIdentification of Identification of phosphorylated phosphorylated sitessitesDIGESTDIGESTIMACIMACMALDI-TOF MS MALDI-TOF MS AnalysisAnalysisPhosphata