肝臟蛋白質(zhì)組計(jì)劃的實(shí)施與毒理學(xué)發(fā)展的機(jī)遇肝臟蛋白質(zhì)組計(jì)劃的實(shí)施與毒理學(xué)發(fā)展的機(jī)遇1（優(yōu)選）肝臟蛋白質(zhì)組計(jì)劃的實(shí)施與毒理學(xué)發(fā)展的機(jī)遇（優(yōu)選）肝臟蛋白質(zhì)組計(jì)劃的實(shí)施與毒理學(xué)發(fā)展的機(jī)遇2曼哈頓原子彈研制計(jì)劃人類基因組計(jì)劃阿波羅登月計(jì)劃人類歷史上的三大科技工程1941.12.6—1945.7.16羅斯福批準(zhǔn),耗資20億美元原子半徑10-10m原子體積10-30m3人體半徑100m人體體積100m3太陽(yáng)系半徑1012m太陽(yáng)系體積1034m31990.10.1-2003.4.23克林頓、布萊爾批準(zhǔn)，耗資30億美元1961.5.25—1969.7.20肯尼迪批準(zhǔn),耗資240億美元曼哈頓原子彈人類基因組計(jì)劃阿波羅登月計(jì)劃人類歷史上的三大科技3人類基因組計(jì)劃開(kāi)創(chuàng)了

“基因組時(shí)代”人類基因組計(jì)劃開(kāi)創(chuàng)了4基因組學(xué)向蛋白質(zhì)組學(xué)“求助”!Nature409:747,15Feb.2001Andnowforproteome

“現(xiàn)在輪到蛋白質(zhì)組”Science291:1221,16Feb.2001ProteomicsinGenomeland“基因組大地中的蛋白質(zhì)組學(xué)”基因組學(xué)向Nature409:747,15Feb.25PROTEINPROTEIN6轉(zhuǎn)錄組一種細(xì)胞、組織或生物體所對(duì)應(yīng)的全套mRNARNA基因組生物體所擁有的全套染色體上的全部基因DNA蛋白質(zhì)組一種細(xì)胞、組織或生物體所對(duì)應(yīng)的全套蛋白質(zhì)PROTEIN功能執(zhí)行體遺傳信息載體轉(zhuǎn)錄組一種細(xì)胞、組織或生物體所對(duì)應(yīng)的全套mRNARNA基因7

基因和蛋白質(zhì)并不存在嚴(yán)格的線性關(guān)系

ORF并不預(yù)示一定存在相對(duì)應(yīng)的功能性基因

mRNA水平并非與蛋白質(zhì)的表達(dá)水平對(duì)應(yīng)翻譯后修飾及同工蛋白質(zhì)(Isoforms)等現(xiàn)象在基因水平無(wú)從表現(xiàn)蛋白質(zhì)與蛋白質(zhì)的相互作用難以在基因水平得以認(rèn)識(shí)基因組與轉(zhuǎn)錄組不能取代蛋白質(zhì)組基因和蛋白質(zhì)并不存在嚴(yán)格的線性關(guān)系基因組與轉(zhuǎn)錄組不能取代蛋8翻譯后修飾相互作用構(gòu)象變化翻譯后拼接移位胞質(zhì)胞核蛋白質(zhì)調(diào)節(jié)的多樣性生命的“萬(wàn)花筒”翻譯后修飾相互作用構(gòu)象變化翻譯后拼接移位胞質(zhì)胞核蛋白質(zhì)調(diào)節(jié)的9蛋白質(zhì)功能的群體性蛋白質(zhì)功能的群體性10繩墻扇茅蛇樹(shù)蛋白質(zhì)作用的整體性繩墻扇茅蛇樹(shù)蛋白質(zhì)作用的整體性11探索生命奧秘的直接對(duì)象蛋白質(zhì)組

生命體的統(tǒng)一性源于基因組生命體的多樣性、復(fù)雜性、功能性、表型源于蛋白質(zhì)組1463-4x1043x109103(1012)

探索生命奧秘的直接對(duì)象蛋白質(zhì)組生命體的統(tǒng)一性源于基因組112

Pteomicsismoreofaconceptthanadefinedtechnology,anditreferstoanyprotein-basedapproachthathasthecapacitytoprovidenewinformationaboutproteinsonagenomewidescale.“Proteomicsincludesnotonlytheidentificationand

quantificationofproteinsbutalsothedeterminationof

their:

localization

modifications

interactions

activities

functionProteomicsseekstoprovidef13報(bào)告提要蛋白質(zhì)組學(xué)產(chǎn)生的時(shí)代背景“國(guó)際人類肝臟蛋白質(zhì)組計(jì)劃”的目標(biāo)與意義蛋白質(zhì)組學(xué)與毒理學(xué)/環(huán)境醫(yī)學(xué)報(bào)告提要蛋白質(zhì)組學(xué)產(chǎn)生的時(shí)代背景14DNA序列圖≠基因圖人類基因組中1/3以上基因未曾確認(rèn)基因確認(rèn)的基本層次--蛋白質(zhì)水平天書(shū)解讀天書(shū)Science291:1221,2001DNA序列圖≠基因圖天書(shū)解讀天書(shū)Science291:1215ItisnecessaryHBPPProteinscore>thresholdMyelomacells85％是針對(duì)目前已知的500種藥靶Protein/peptideidentificationKEEpSEEpSDDDMGFGLFD重大疾病發(fā)生發(fā)展機(jī)制Subcellular:Mitochondria,Golgi,PM,Nucleus,EvaluationoftechnologiesESEALPEKEGEELGEGERPEEDAAALELpSpSDEAVEVEEVIEESRMS/MSAnalysisandDatabaseSearchingThefirstphase:2004-2005組織的活力依賴于高能鍵的連續(xù)生成。物質(zhì)——代謝（活性分子的合成、肯尼迪批準(zhǔn),耗資240億美元Evaluationoftechnologies3,Washington Organizer:LauraBereta肝臟蛋白質(zhì)組計(jì)劃的實(shí)施與毒理學(xué)發(fā)展的機(jī)遇肝炎向肝癌的惡性轉(zhuǎn)化

難以遏制全面揭示重大疾病發(fā)生發(fā)展機(jī)制的基礎(chǔ)蛋白質(zhì)組單基因病—遺傳病多基因病—腫瘤等

重大疾病發(fā)生發(fā)展機(jī)制單一基因單一蛋白基因群蛋白質(zhì)群轉(zhuǎn)錄組蛋白質(zhì)組????Itisnecessary全面揭示重大疾病發(fā)生發(fā)展機(jī)制的16發(fā)現(xiàn)大量新型藥靶與新藥的源泉蛋白質(zhì)組最重要的疾病100-150每種疾病相關(guān)的基因10每個(gè)基因相關(guān)的蛋白質(zhì)3–10藥靶蛋白總數(shù)

3000-15,000 現(xiàn)有藥物約2000種85％是針對(duì)目前已知的500種藥靶6-30倍藥靶有待發(fā)現(xiàn)發(fā)現(xiàn)大量新型藥靶與新藥的源泉蛋白質(zhì)組最重要的疾病17啟動(dòng)人類蛋白質(zhì)組計(jì)劃勢(shì)在必行！探索人體生命奧秘的直接對(duì)象全面揭示重大疾病發(fā)生發(fā)展機(jī)制的基礎(chǔ)發(fā)現(xiàn)大量新型藥靶與新藥的源泉

人類基因組序列及基因注釋人類蛋白質(zhì)組VIP:VeryImportantProteome啟動(dòng)人類蛋白質(zhì)組計(jì)劃探索人體生命奧秘的直接對(duì)象全面揭示重大疾18人類蛋白質(zhì)組計(jì)劃的

主要科學(xué)目標(biāo)驗(yàn)證人類基因組計(jì)劃推測(cè)的基因，注釋基因組闡明蛋白質(zhì)組的調(diào)控模式并與轉(zhuǎn)錄組進(jìn)行對(duì)比建立蛋白質(zhì)群/組裝體，蛋白質(zhì)復(fù)合物或蛋白質(zhì)機(jī)器,即連鎖圖建立人體生理學(xué)、病理學(xué)的蛋白質(zhì)組基礎(chǔ)人類蛋白質(zhì)組計(jì)劃的

主要科學(xué)目標(biāo)驗(yàn)證人類基因組計(jì)劃推測(cè)的基因191463-4x1043x109103(1012)

基因組計(jì)劃蛋白質(zhì)組計(jì)劃1463-4x1043x109103(1012)20肝臟蛋白質(zhì)組計(jì)劃的實(shí)施與毒理學(xué)發(fā)展的機(jī)遇講課課件21Bodyfluidmostaccessibleforbiomarkerdiscoveryinanimalandhumans.LiverKidneyOrganBodyFluidBrainImmuneOrgans/VesselsIntestineOtherTissuesCSFFecesEndocrine/ParacrineSecretionsBileUrineAir/BALFLymphLungsEyesSkinOralCavitySweatSalivaTearsVenousBloodArterialBloodSerum10%90%SerumProteinsNon-InformativeAbundantProteinsi.e.albumin,IgG,etc.InformativeLowAbundanceProteinsEnrichmentStrategiesforInformativeSerumProteins:SELDI

SerumImmunosubtraction

Bodyfluidmostaccessiblefor22人類肝臟蛋白質(zhì)組計(jì)劃里程碑蛋白表達(dá)譜蛋白連鎖圖亞細(xì)胞定位圖修飾譜人類基因組計(jì)劃

里程碑遺傳圖物理圖序列圖人類肝臟蛋白質(zhì)組蛋白表達(dá)譜人類基因組計(jì)劃

里程碑遺傳圖23InitiativesofHumanProteomeProject(HPP)HPPPHumanPlasmaProteomeProject,USAHLPPHumanLiverProteomeProject,ChinaPSIProteomicsStandardsInitiativeUKHBPPHumanBrainProteomeProject,GermanyHAIHumanAntibodyInitiativeSwedenInitiativesofHumanProteome24Whyisliver?wwwww.HLPP.HUPO為何研究肝臟?Whyisliver?wwwww.HLPP.HUPO為何25肝臟功能的多樣性與重要性能量——能量轉(zhuǎn)換、儲(chǔ)存物質(zhì)——代謝（活性分子的合成、毒性分子的分解）信息——信息分子的合成與分泌肝臟功能的多樣性與重要性能量——能量轉(zhuǎn)換、儲(chǔ)存26肝臟為其它組織提供能量—可氧化底物組織的活力依賴于高能鍵的連續(xù)生成。肝臟產(chǎn)生大部分脂肪酸，后者是禁食狀態(tài)下能量的基本來(lái)源。肝臟是給食狀態(tài)下由過(guò)剩糖合成脂肪酸的主要部位。肝臟為其它組織提供能量—可氧化底物組織的活力依賴于高能鍵的連27藥物毒物營(yíng)養(yǎng)物生物異源物生物轉(zhuǎn)化藥物毒物營(yíng)養(yǎng)物生物異源物生物轉(zhuǎn)化28RongZeng(ShanghaiInstituteforBiologicalSciences)重大肝病的發(fā)生發(fā)展無(wú)法歸咎于少數(shù)幾個(gè)基因或造血系統(tǒng)的發(fā)育必需肝臟的“培育”GUI，WebServices,APIOtherTissuesPhosphopeptideConfirmation增強(qiáng)疾病預(yù)防診斷與治療InitiativeCoordinationAntibodies了解化學(xué)毒物的代謝途徑FuchuHe:Mar22,2003(ActionPlan);Dr.Meyer,Ueffing)Generateanintegrativeapproachleadingtoacomprehensivefunctionalmapoftheliver

Expandliverproteometoits“PHYSIOME”and“PATHOME”todramaticallyacceleratethedevelopmentofdiagnostics,preventionandtherapeuticstowardsitsdiseases

Developstandardoperatingprocedures(SOPs)forotherHUPOInitiatives世界上每年有100萬(wàn)新發(fā)原發(fā)性肝癌病人，其中40-50%在我國(guó)10,EBI/London Organizer:RolfApweilerProteinisolationandpurificationbylectin(Glycosylation)PrimaryanalysisofFrenchlivertissues蛋白質(zhì)組學(xué)產(chǎn)生的時(shí)代背景肝臟功能的多樣性與重要性HLPPAntibodyBankatPilotPhase化學(xué)多樣性向體內(nèi)的生物轉(zhuǎn)化體系提出嚴(yán)峻挑戰(zhàn)1.2×107種以上的化學(xué)物(CAService’slist)以每周約8000種的速率增加常用化學(xué)物在63,000種以上大約11,500種，作為食物或藥物制劑添加物攝入其余的50,000種，為潛在的環(huán)境污染物RongZeng(ShanghaiInstitute29人類發(fā)育過(guò)程中造血組織的興替造血系統(tǒng)的發(fā)育必需肝臟的“培育”人類發(fā)育過(guò)程中造血組織的興替造血系統(tǒng)的發(fā)育必需肝臟的“培育”30合成大多數(shù)循環(huán)血漿蛋白合成和分泌血漿蛋白是肝臟實(shí)質(zhì)細(xì)胞——肝細(xì)胞的獨(dú)有功能（肝細(xì)胞占肝臟總細(xì)胞數(shù)的60%及肝臟質(zhì)量的約80%）最有特征的血漿蛋白是白蛋白，在大多數(shù)哺乳動(dòng)物中占總血漿蛋白的55-60%凝血酶、抗凝因子、溶栓酶等合成大多數(shù)循環(huán)血漿蛋白合成和分泌血漿蛋白是肝臟實(shí)質(zhì)細(xì)胞——肝31肝臟再生機(jī)體再生能力最強(qiáng)的器官終生保持旺盛的再生能力肝臟再生機(jī)體再生能力32肝臟中包含的“組”(-ome)Metabolicgenomics→Metabolome

(代謝組)Energygenomics→Energome

(能量組)Pharmacogenomics→Pharmacome

(藥理組)Toxicogenomics→Toxicome

(毒理組)Regeneratinggenomics

→Regenerome

(再生組)肝臟中包含的“組”(-ome)Metabolicgenom33全球及中國(guó)肝炎的流行病學(xué)統(tǒng)計(jì)

全球:3.5億攜帶者中國(guó):1.8億攜帶者死亡:23萬(wàn)人/年疾病負(fù)擔(dān):500億元人民幣治療手段:抗病毒,保肝降 酶,抗纖維化,免疫治療等缺點(diǎn):病毒易反彈,病 情易反復(fù)全球及中國(guó)肝炎的流行病學(xué)統(tǒng)計(jì)34肝炎向肝癌的惡性轉(zhuǎn)化

難以遏制全世界現(xiàn)有3.5億慢性乙肝病毒（HBV）攜帶者，占世界人口的5%，亞洲和非洲HBV攜帶率為8-15%

HBV攜帶者中50-70%病毒復(fù)制活躍，為慢性乙肝

慢性乙肝病人肝硬化發(fā)生率為2-20%，代償性肝硬化發(fā)展成失代償性肝硬化為20-23%，發(fā)展成肝癌的占6-15%中國(guó)的慢性乙肝病人中，約25-40%最終將死于肝硬化或合并肝癌HBV攜帶者最終死于相關(guān)肝病的危險(xiǎn)性，男性為50%，女性為15%

肝炎向肝癌的惡性轉(zhuǎn)化

難以遏制全世界現(xiàn)有3.5億慢性乙肝病毒35全球:100萬(wàn)人/年中國(guó):50萬(wàn)人/年死亡:約45萬(wàn)人/年疾病負(fù)擔(dān):400億元人民幣治療手段:手術(shù)切除(只有 15%-20%)治療效果:術(shù)后易轉(zhuǎn)移,易復(fù) 發(fā),五年存活率 40-50%全球及中國(guó)肝癌的流行病學(xué)統(tǒng)計(jì)全球及中國(guó)肝癌的流行病學(xué)統(tǒng)計(jì)36原發(fā)性肝癌的治療水平亟待突破世界上每年有100萬(wàn)新發(fā)原發(fā)性肝癌病人，其中40-50%在我國(guó)

我國(guó)原發(fā)性肝癌發(fā)病率和死亡率均位居第二位

近20年來(lái)我國(guó)肝癌的發(fā)病率上升近40%原發(fā)性肝癌一般起病較隱匿，早期缺乏典型臨床表現(xiàn)，初診大多已屬中晚期

初診肝癌病人中，只有15-20%的病人具備手術(shù)條件這些病人術(shù)后5年生存率僅為40-50%

肝癌是致死率最高的惡性腫瘤之一原發(fā)性肝癌的治療水平亟待突破世界上每年有100萬(wàn)新發(fā)原發(fā)性肝37重大肝病的發(fā)生發(fā)展無(wú)法歸咎于少數(shù)幾個(gè)基因或蛋白質(zhì)所呈現(xiàn)的功能狀態(tài)和信號(hào)通路從蛋白質(zhì)組層面上“全景式”揭示肝臟疾病的生理病理機(jī)制是解決重大肝病的根本出路肝炎病毒肝臟/肝細(xì)胞肝炎肝硬化/纖維化原發(fā)肝癌肝癌轉(zhuǎn)移多因素、多步驟的發(fā)病機(jī)制重大肝病的發(fā)生發(fā)展無(wú)法歸咎于少數(shù)幾個(gè)基因或從蛋白質(zhì)組層面上“38肝臟是人類蛋白質(zhì)組計(jì)劃的首批目標(biāo)！肝臟是人類蛋白質(zhì)組計(jì)劃的首批目標(biāo)！39化學(xué)多樣性向體內(nèi)的生物轉(zhuǎn)化體系提出嚴(yán)峻挑戰(zhàn)增強(qiáng)疾病預(yù)防診斷與治療Antibodycharacterizationandapplication肯尼迪批準(zhǔn),耗資240億美元GeneticsInfluenceEnvironment物質(zhì)——代謝（活性分子的合成、Proteinscore≥59(Rank1forthespot)GUI，WebServices,APISomesimilarproteinswereidentified(notproteingroup),suggestingthecriteriafordistinguishingsimilarproteinsshouldbemorestrict.EstablishtheSOPsfortheblackracialpeople`sliversample了解化學(xué)毒物的代謝途徑EvaluationoftheSOPsforthesubcellularfractionationADpSGEGDFLAEGGGVRGSLApSLDpSLRKIdentificationofphosphorylatedsites5millionUSdollarsGFP-fusedmutantHUPO2ndWorldCongress,Montreal,Oct.Prometheusstolefirefromthegodsandgaveittomortals.PrometheusBoundHeraclesLiberatePrometheusHLPP—ModernPrometheusMythPrometheus

LiverEagleLiverdiseases

HeraclesHLPP化學(xué)多樣性向體內(nèi)的生物轉(zhuǎn)化體系提出嚴(yán)峻挑戰(zhàn)Prometheu40人類肝臟蛋白質(zhì)組計(jì)劃

HumanLiverProteomeProject(HLPP)國(guó)際HLPP共同體人類肝臟蛋白質(zhì)組計(jì)劃

HumanLiverProteom41Generateanintegrativeapproachleadingtoacomprehensivefunctionalmapoftheliver

Expandliverproteometoits“PHYSIOME”and“PATHOME”todramaticallyacceleratethedevelopmentofdiagnostics,preventionandtherapeuticstowardsitsdiseases

Developstandardoperatingprocedures(SOPs)forotherHUPOInitiativesVISIONGenerateanintegrativeapproa42HUPOWorkshop,Bethesda,April28-29,2002

BroadinterestsandsupportsforinitiatingHLPPHUPOLiverProteomeWorkshop,Beijing,October22-24,2002

GettingconsensusviewforscientificobjectivesofHLPPHUPO1stWorldCongress,Versailles,November21-24,2002

Co-chairsofHLPP:Drs.FuchuHe,JohnBergeronandChristianBréchotHLPPPlanningCommittee: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,July17-18,2003NIHparticipatingHUPO2ndWorldCongress,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.,2005MISIONHUPOWorkshop,Bethesda,April43表達(dá)譜修飾譜定位圖連鎖圖樣品庫(kù)抗體庫(kù)數(shù)據(jù)庫(kù)HLPP的科學(xué)目標(biāo)---肝臟蛋白質(zhì)組的“太陽(yáng)系”3-D圖ORF組表達(dá)譜修飾譜定位圖連鎖圖樣品庫(kù)抗體庫(kù)數(shù)據(jù)庫(kù)HLPP的科學(xué)目標(biāo)44肝臟蛋白質(zhì)組與

肝臟生理、病理的銜接肝臟蛋白質(zhì)組代謝組毒理組藥理組再生組肝炎組肝癌組能量組肝臟蛋白質(zhì)組與

肝臟生理、病理的銜接肝臟蛋白質(zhì)組代謝組毒理組45作為分泌器官，肝臟合成大多數(shù)的循環(huán)血漿蛋白

肝臟轉(zhuǎn)錄組“肝臟蛋白質(zhì)組計(jì)劃”和“血漿蛋白質(zhì)組計(jì)劃”的整合肝臟蛋白質(zhì)組血漿蛋白質(zhì)組?作為分泌器官，肝臟合成大多數(shù)的“肝臟蛋白質(zhì)組計(jì)劃”和肝臟蛋白46基因組肝臟蛋白質(zhì)組肝臟轉(zhuǎn)錄組基因組、轉(zhuǎn)錄組和蛋白質(zhì)組的集成證實(shí)推測(cè)基因?qū)Ρ日{(diào)控模式

基因組肝臟肝臟基因組、轉(zhuǎn)錄組和蛋白質(zhì)組的集成證實(shí)推測(cè)基因?qū)Ρ?7重要功能蛋白質(zhì)肝病藥物靶標(biāo)肝病診斷標(biāo)志物人類肝臟蛋白質(zhì)組重要功能蛋白質(zhì)肝病藥物靶標(biāo)肝病診斷標(biāo)志物人類肝臟蛋白質(zhì)組48肝炎病毒肝臟/肝細(xì)胞感染肝炎肝硬化/纖維化原發(fā)肝癌肝癌轉(zhuǎn)移重大肝病預(yù)警、診斷、預(yù)防、治療的關(guān)鍵環(huán)節(jié)“東亞病夫”“肝病大國(guó)”1億多肝炎病毒攜帶者1000億多/年醫(yī)療費(fèi)用肝炎病毒肝臟/肝細(xì)胞感肝炎肝硬化/纖維化原發(fā)肝癌肝癌轉(zhuǎn)移重大49SOPsSpecimenBankingPlatformsWorkingteamGlobalCollaborationExpressionProfileORFeomeAntibodiesBioinformatics…ModificationProfileSubcellularLocalizationProtein-ProteinInteractionAntibodiesBioinformaticsIntegrateproteomewithtranscriptomeCompareproteomeswithinhealthanddiseasedliverTimeLinesPilotPhase(2003-2005)PhaseII(2006-2010)SOPsModificationProfileTime50OutlineBackgroundProgressoftheHLPP-ProgressreportonexpressionprofilingInitiationoftheCNHLPPNextworkOutlineBackground51Progress-ExpressionprofilingEvaluationoftechnologiesBioinformaticsset-upSamplingandpreparationPreviewwithhumanfetalliverPrimaryanalysisofFrenchlivertissuesProgress-Expressionprofilin52ProgressEvaluationoftechnologiesBioinformaticsset-upSamplingandPreparationPreviewwithhumanfetalliverPrimaryanalysisofFrenchlivertissuesProgressEvaluationoftechnolo53Majorconclusionsfromthevaluationoftechnologies–16standardproteins/7labsHighmolecularweightandlowabundanceproteinsarelessdetectableby2DE.Proteinswithonlythreeorderofmagnitudecouldbeidentifiedin2DE,butproteinswithfourorderofmagnitudecouldbeidentifiedinshotguntechnology.Morepeptides(redundant)wereidentifiedforhighMWproteinsinshotguntechnology.Somesimilarproteinswereidentified(notproteingroup),suggestingthecriteriafordistinguishingsimilarproteinsshouldbemorestrict.Majorconclusionsfromtheval54ProgressEvaluationoftechnologiesBioinformaticsset-upSamplingandPreparationPreviewwithhumanfetalliverPrimaryanalysisofFrenchlivertissuesProgressEvaluationoftechnolo55HLPPExperimentalDatabaseMWSDataClient…GUI，WebServices,APIFilesInterchange,DMBSDumpDatasynchronizeDatasubmissionExperimentreports,data,files…

HLPPDataMirrorMWSDataClientMWSDataClientMWSDataClientMWSDataClientMWSServerAdministratorHLPPDataMirrorSubmissionPackagesRepositoryHLPPProjectManagementDatabaseHLPPProjectManagementSystemHLPPParticipatingLaboratoriesHLPPDataCenterTheHLPPDataManagementSystem

ArchitectureBasedonMyWorkSpaceSystem(MWS)HLPPExperimentalMWSDataCli56CurrentStandarddraftofHLPPSampleInformationProteinextractionConcentrationMeasurementSeparationGel-based2DEGel1D-IEFGel1D-SDSLC-basedRPLCSCXLCSAXLCSECTrapDesaltingDigestionIonsourceMALDIAutoflexUltraflexABI4700ESIQstarQTofMicroLCQLTQMassspectrometryTOFTOFTOFUltraflexABI4700QTOFQstarQTofMicroITMSLCQLTQPeaklistgenerationABI4700QStarAutoflexTurboSequestMasslynxFlexAnalysisPeaklistpreprocessingGPSpeaklistpreprocessProtein/peptideidentificationMascotSequestProteinlistgenerationAutoflexGPSBiotoolsQstarBuildSummaryDTASelectBioworksAutoQuestCurrentStandarddraftofHLPP57Proteinidentificationisbasedontheanalysisofpeptidesgeneratedbyproteolyicdigestion.Whenthedetectedpeptidesmatchmanyproteinsandcannotidentifyauniqueprotein,theresultwillbepresentedintheformofagroupofpossibleproteins.

DefinitionofProteinGROUP

Proteinidentificationisbase58CriteriaforproteinidentificationIontrapXCorr(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)Criteriaforproteinidentific59ProgressEvaluationoftechnologiesBioinformaticsset-upSamplingandpreparationPreviewwithhumanfetalliverPrimaryanalysisofFrenchlivertissuesProgressEvaluationoftechnolo60EvaluationoftheSOPsforthesubcellularfractionationObject:FindtheoptimummethodofpretreatmentSample:LivertissuesofC57miceSubcellular:Mitochondria,Golgi,PM,Nucleus,ER,CytoplasmSamplepretreatmentFreshtissuesFrozenhomogenisedtissuesFrozentissuesEvaluationoftheSOPsforthe61Purity(Westernblotting):Freshtissues>frozenhomogenizedtissues>frozentissuesTheyieldofproteins:Freshtissues>frozenhomogenizedtissues>frozentissuesIntegrity(TEM):Freshtissues>frozenhomogenizedtissuesSummaryPurity(Westernblotting):Fre62ProgressEvaluationoftechnologiesBioinformaticsset-upSamplingandPreparationPreviewwithhumanfetalliverPrimaryanalysisofFrenchlivertissuesProgressEvaluationoftechnolo63ItisagiantambitiousobjectiveandgivesagreatchallengetoembryonicproteomicsItisnecessarytocarryoutapreviewbeforetheformallaunchingofthisprojectScience302:1316,Nov.21,2003Nature425:441,Oct.2,2003Itisagiantambitiousobject64AverageThroughputofthePlatform2DESystem

12Gel/3DaysAuto-SpotDigestSystem

1152spots/DayMSandMS/MSSystem

500-1000Spots/DayAverageThroughputofthePlat65TheflowchartofCCPITfortheproteinexpressionprofileofhumanfetalliverTheflowchartofCCPITforthe66Theareascalesbeforeandafterisoformprocessingingroupandproteinlevelsrespectively.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:

1258Theareascalesbeforeand67ACBChromosomaldistributionofHFLproteome-encodinggenesACBChromosomaldistributionof68FunctionalModulesinplasmamembraneFunctionalModulesinplasmam69ProgressEvaluationoftechnologiesBioinformaticsset-upSamplingandPreparationPreviewwithhumanfetalliverPrimaryanalysisofFrenchlivertissuesProgressEvaluationoftechnolo70

ReferenceLabsforHLPPExpressionProfilingProject

FuchuHe&XiaohongQian(BeijingInstituteofRadiationMedicine)RongZeng(ShanghaiInstituteforBiologicalSciences)PengyuanYang(FudanUniversity)SiqiLiu(BeijingGenomicsInstitute,ChineseAcademyofSciences)ReferenceLabsforHLPPExpre71LauraBeretta(FHCRC,USA)RichardJ.Simpson(RoyalMelbourneHospital,Australia)

AngelikaGorg(TechnischeUniversitatMunchen,Germany)MarkBaker/JunhongSun(APAF,Australia)LauraBerettaRichardJ.Simps72OutlineBackgroundProgressoftheHLPPInitiationoftheCNHLPPNextworkOutlineBackground73CNHLPPOverviewThefirstphase:2004-2005Fundingfromcentralgovernment:10millionUSdollars ChineseMOST:jointlyfundedbyNationalProgramon KeyBasicResearchProjects(973),NationalHigh-tech R&DProgram(863),andNationalKeyTechnologiesR&D Programmeininthefirstphase.LocalandInstitutionalfunding:3.5millionUSdollars8subprojects70institutes,universities,andcompaniesinvolvedLong-termsupportasanationalproject(2006~2020)CNHLPPOverviewThefirstphase74CelebrationofCNHLPPOfficialLaunching(BeijingInstituteofRadiationMedicine)CelebrationofCNHLPPOfficial75KeylabsBeijingInstituteofRadiationMedicineKeylabsBeijingInstituteof76Consultingcommittee(5)ORFeome&PPISubproject,ZeguangHanChairFuchuHeHeadquartersBPRCModificationProfilesubproject,YunCaiExpressionProfileSubproject,XiaohongQianStructuralBiologySubproject,WeiminGongBioinformaticssubproject,YixueLiNewTechnologysubproject,YukuiZhangAntibodysubproject,Qi-HongSunLocalizationMappingSubproject,XueminZhangConsultingcommitteeORFeome&77Morethan10workshopshavebeenheldMorethan1078Briefprogressofsomesubprojects

ProteinmodificationProteinlocalizationProteinstructureNewproteomicstechnologiesBriefprogressofsomesubproj79AnalysisofproteinphosphorylationbycombinationofIMAC,PhosphatasewithBiologicalMassSpectrometrym/zPeptidemixtureProteinsCandidatephosphopeptidesIdentificationofphosphorylatedsitesDIGESTIMACMALDI-TOFMSAnalysisPhosphatasedigestMS/MSAnalysisandDatabaseSearchingm/zPhosphopeptideConfirmationm/zMetastableionsofphosphopeptidesAnalysisofproteinphosphoryl80O60716

GSLApSLDpSLRKP15924GLPSPYNMSpSAPGpSRO75379NLLEDDpSDEEEDFFLRRApSpSKGGGGYTC*QSGSGWDEFTKO95210HSpSWGDVGVGGSLKP16157RDpSRDVDEEKELLDFVPKP02671ADpSGEGDFLAEGGGVRP35221TPEELDDpSDFETEDFDVRP02730

YQpSSPAKPDSSFYKQ13813WRpSLQQLAEERYQSSPAKPDSSFpYKQ15019IYHLPDAEpSDEDEDFKEQTRYQSSPAKPDSpSFYKQ16828SNIpSPNFNFMGQLLDFERRYQSSPAKPDpSSFYKQ99959RLEISPDpSpSPERAHYTHSDYQYSQRP04792GPpSWDPFRLPEEWSQWLGGSSWPGYVRPLPPAAIEpSPAVAAPAYSRQ9H3Z4SLpSTSGESLYHVLGLDKTr:O95810SLEETLHTVDLpSpSDDDLPHDEEALEDpSAEEKVEESRP05386KEEpSEEpSDDDM*GFGLFDKEEpSEEpSDDDMGFGLFDTr:Q8NEN5pSQpSLPTTLLSPVRP08559YHGHpSMSDPGVSYRTr:Q9HAP4ESEALPEKEGEELGEGERPEEDAAALELpSpSDEAVEVEEVIEESRP11277TpSPVSLWSRLpSpSSWESLQPEPSHPYTr:Q9NQA7WLDEpSDAEMELRIdentifiedphosphopeptidesequencesfromHFLO60716GSLApSLDpSLRKP15924GLPS81Proteinisolationandpurificationbylectin(Glycosylation)Proteinisolationandpurifica82Co-localizationandlocomotionoftheGFPfusionproteinandDsRedDsRed-MemGreenmergeGFPGFP-fusedPrTNF-treatedGFP-fusedmutantTNF-treatedGFP-fusedPrCo-localizationandlocomotion83StructuralproteomicsCloned900genesfromliverExpressedproductsofmorethan100genesDeterminedthestructuresof3proteinsbyX-rayDeterminedthestructuresof2proteinsbyNMRStructuralproteomicsCloned9084Diphosphoglyceratemutase(DPGM)X-rayProgrammedcelldeath5(PDCD5)NMRDiphosphoglyceratemutaseX-ray85Beijing(National)ProteomeResearchCenterChineseProteomeDevelopmentCenterBeijing(National)ProteomeCh86TheAntibodyBankofHumanLiverProteomeProjectEstablishment,characterization,andapplicationofantibodiesagainsthumanliverproteinsQi-HongSunBeijingInstituteofRadiationMedicine(BIRM)BeijingProteomeResearchCenter(BPRC)TheAntibodyBankofHumanLiv87AntigensMyelomacellsHybridomacellsmAbsDiagnosticsTherapeuticsProteomicsResearchProfilingIdentificationQuantificationLocalizationModificationInteractionFunctionLargeantibodycollectionforproteomicsAntigensMyelomacellsHybridoma88AntibodyDatabaseAntibodyBankSamples5000liverproteinsHLPPAntibodyBankAntibodychipsAntibodyDatabaseAntibodyBank8910%ofHLPPAntibodyBankStandardsanddatabaseofHUPOHLPP/HAIMicroarrayandotherantibody-basedtechnology90%10%HLPPAntibodyBankatPilotPhase10%ofHLPPAntibodyBank90%190AntigenpreparationFractionatedliverproteins-Unknown/native/multi-antigensRecombinantproteinsSynthesizedpeptidesAntibodygenerationHybridomacelllines–mAbs(murine/rabbit)Polyclonalantibodies–IgG(Rabbit)/orIgY(chicken)AntibodycharacterizationandapplicationClassandsubclass,ELISA,IH,WB,andIPAntibodymicroarrayandotherantibody-basedtechnologyGeneralApproachAntigenpreparationGeneralAp91AdultFetalDiseasedIHcharacterizationofmAbsagainsthumanliverproteinsAdultFetalDiseasedIHcharacter92ProgresssummaryofHLPPAntibodyBankingProject

AntigenpreparationforimmunizationFractionatedliverproteins–nucleus,membrane,microsome,mitochondrial,cytosolic,andplasmaproteinsPurifiedrecombinantproteinantigens–215Synthesizedpeptidesfromliver-specificproteins–44AntibodypreparationandcharacterizationEstablishedhybridomacelllines–1085CharacterizedmAbs–862formorethan100differentproteinsRabbitpAbs–50AntibodyApplication

Depletionofhigh-abundantproteinsImmunohistochemistryIsolationofproteincomplexesProgresssummaryofHLPPAntib93LabsofHLPPAntibodyBankingProjectNational(China):Qi-HongSun/JianenGao,MingLi,MinZhao,DalingZhu,ChaonengJi,GangCheng,ZhinanChen,BoquanJing,JianWang/LinWu,XiaoningWang/YingLin,ZhengLi,JinliangYang,andJieTang.Internationalcollaboration:HUPO-HAI(Dr.Uhlen)FHCRC/NCI-IBDC(Dr.Lee)GermanSystemsBiologyProject(Drs.Meyer,Ueffing)LabsofHLPPAntibodyBanking94Workingplan

Antibodydatasheet–ElectronicsubmissionformAntibodylist–Website/HLPPAntibodydatabaseAntibodydemands/applicationsAntibodyqualitycontrolAntibodyexchange,collection,anddistributionScaleofantibodybankAntibody-basedtechnologyInternationalcollaborationWorkingplan95肝臟蛋白質(zhì)組計(jì)劃的實(shí)施與毒理學(xué)發(fā)展的機(jī)遇講課課件96OutlineBackgroundProgressoftheHLPPInitiationoftheCNHLPPNextworkOutlineBackground97ResearchprojectsDistributetheChinesespecimenstoreferencelabsoutsideChinaEstablishtheSOPsfortheblackracialpeople`sliversampleComprehensivelyanalyzethedataoftheproteinsexpressingprofileoftheFrenchspecimensEstablishtheSOPsfortheprotein-proteininteractionsandORFeomeInitiatevirtuallydiseasedliverproteomesubprojectInitiatevirtuallyproteomicmodificationandlocalizationsubprojectsResearchprojects98CoordinationContinuetodiscussthecooperationwithotherinitiatives(e.g.MRPP,HPPP,PSI,HAI,etc.)orotherprogramme(e.g.SystemsBiologyProjectofGermany)EstablishtheheadquartersoftheHLPPContinuetopropagandizetheHLPPtothepublicandprivatedomainsStriveformorefinancialsupportsfrommoreresourcesCoordinationContinuetodiscus99HLPPworkshopsJun.3,Washington Organizer:LauraBeretaJun.10,EBI/London Organizer:RolfApweilerAug.27,Munich Organizer:FuchuHeHLPPworkshops100報(bào)告提要蛋白質(zhì)組學(xué)產(chǎn)生的時(shí)代背景“人類蛋白質(zhì)組計(jì)劃”的目標(biāo)與意義蛋白質(zhì)組學(xué)與毒理學(xué)/環(huán)境醫(yī)學(xué)報(bào)告提要蛋白質(zhì)組學(xué)產(chǎn)生的時(shí)代背景101主要作用揭示環(huán)境因素的致病機(jī)制了解化學(xué)毒物的代謝途徑解析微生物蛋白質(zhì)組組成增強(qiáng)疾病預(yù)防診斷與治療主要作用揭示環(huán)境因素的致病機(jī)制102Genetics

Influence

Environment

GenomicsTranscriptomics2-DE/MSICAT-LC/MSInformationFlowDNAm-RNAProteinProtein

species2Proteinspecies2ProteinspeciesnRibosomePTMsProtein

species1Protein

species1Protein

species2ProteomicsGenetics103基因（組）環(huán)境因素蛋白質(zhì)（組）疾病揭示環(huán)境因素的致病機(jī)制蛋白質(zhì)組學(xué)系統(tǒng)研究機(jī)體的蛋白質(zhì)變化揭示環(huán)境因素對(duì)人體的致病機(jī)制生物因素化學(xué)因素物理因素基因（組）環(huán)境因素蛋白質(zhì)（組）疾病揭示環(huán)境因素的致病機(jī)104ThemissionoftheintramuralProteomicsprogramwithintheNationalCenterforToxicogenomics(NCT)istoidentifykeyproteinsandpathwaysinvolvedintheresponsetoenvironmentaltoxicantsandstressors.Proteomictechnologiesarebeingusedtoassessglobalchangesinproteinexpressioninordertoidentifybiomarkersofchemicalexposureandenvironmentallyrelateddiseases.NationalCenterforToxicogenomics(NCT)Themissionoftheintramural105Mission:Toidentifykeyproteinsandpathwaysresponding

toenvironmentaltoxicantsanddiseaseusing

global

proteinanalysis.Goals:Toidentifytoxicantexposureorenvironmentaldiseaserelatedto: 1)ProteinBiomarkers:TargetTissueandSerum 2)ConductparallelMicroarray&Proteomicstudies 3)Biochemicalpathways, protein-proteininteractions,PTMs 4)Fostertechnicalinnovationsforglobalproteinanalysis. Mission:Toidentifykeyprote106StrategiesforAnalysisofToxicoproteomicDataStrategiesforAnalysisofTox107藥物毒物營(yíng)養(yǎng)物生物異源物生物轉(zhuǎn)化藥物毒物營(yíng)養(yǎng)物生物異源物生物轉(zhuǎn)化108了解化學(xué)毒物的代謝途徑蛋白質(zhì)組學(xué)代謝網(wǎng)絡(luò)化學(xué)因素代謝物了解化學(xué)毒物的代謝途徑蛋白質(zhì)組學(xué)代謝網(wǎng)絡(luò)化學(xué)因素代謝物109ThemetabolismpathwaysofLiverproteomeThemetabolismpathways110解析微生物蛋白質(zhì)組組成解析微生物蛋白質(zhì)組組成111Theroutetoanewmed