肝脏蛋白质组计划的实施与毒理学发展的机遇课件1.ppt

1、肝脏蛋白质组计划的实施与毒理学发展的机遇（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

2、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”基因组学向基因组学向蛋白质组学蛋白质组学“求助求助”!Nature 409:747,15 Feb.2001 And now for proteome“现在轮到蛋白质组现在轮到蛋白质组”Science 291:1221,16 Feb.2001Proteomics in Genomeland“基因组大地中的蛋白质组学基因组大地中的蛋白质组学”PROTEINPROTEIN转录组转录组一种细胞、组织或生物体所对应的全套全

3、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 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

4、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质调节的多样性蛋白质调节的多样性生命的生命的“万花筒万花筒”蛋白质功能的群体性蛋白质功能的群体性Antibody databaseMore peptides(redundant)were identified for high MW proteins in shotgun technology.Nature 409:747,15 Feb.8 subprojectslocalization1152 spo

5、ts/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 technol

6、ogy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探索生命奥秘的直接对象探索生命奥秘的直接对象蛋白质组蛋白质组 生命体的统一性源于基因组生命体的统一性源于基因组 生命体的多样性、复杂性、生命体的多样性、复杂性、功能性、表型源于蛋白质组功能性、表型源于蛋白质组1463-4 x 1043 x 109103(1012)n Proteomics seeks to provide functional information for all proteins.n proteomics is more of a concept than a defined technology,and i

7、t 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 f

8、unction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”的目标与的目标与意义意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DNA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Science 291:1221,2001全面揭示重大疾病发生发展机制的基础全面揭示重大疾病发生发展机制的基础蛋白质组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

9、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?Rabbit 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 t

10、issuesHLPP 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 technologyESEALPEKEGEE

11、LGEGERPEEDAAALELpSpSDEAVEVEEVIEESR发现大量新型药靶与新药的源泉发现大量新型药靶与新药的源泉蛋白质组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 3 10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探索人体生命奥秘的直接对象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发现大量

12、新型药靶与新药的源泉 人类基因组序列及基因注释人类基因组序列及基因注释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类基因组计划推测的基因，注释基因组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建立蛋白质群/组装体，蛋白质复合物或蛋白质组装体，蛋白质复合物或蛋白质机器机器,即连锁图即连锁图建立人体生理学、病理学的蛋白质组基础建立人体生理学、病理学的蛋白质组基础1463-4 x 1043 x 109103(1012)基因组计划基因组计划蛋白质组计划蛋白质组计划Body fluid most

13、 accessible for biomarker discovery in animal and humans.LiverKidneyOrganBody FluidBrainImmune Organs/VesselsIntestineOther TissuesCSFFecesEndocrine/Paracrine SecretionsBileUrineAir/BALFLymphLungsEyesSkinOral CavitySweatSalivaTearsVenous BloodArterial BloodSerum10%90%Serum ProteinsNon-Informative Ab

14、undant Proteins i.e.albumin,IgG,etc.Informative Low Abundance ProteinsEnrichment Strategies for Informative Serum Proteins:SELDI Serum Immunosubtraction 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谱蛋白表达谱蛋白连锁图蛋白连锁图亚细胞定位图亚细胞定位图修饰谱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遗传图物理图物理图序列图序列图Initiatives of Human Proteome Project(HPP)HPP

15、PHuman Plasma ProteomeProject,USAHLPPHuman Liver Proteome Project,ChinaPSIProteomics Standards Initiative UK HBPP Human Brain Proteome Project,GermanyHAIHumanAntibodyInitiativeSwedenAntibody 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

16、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 ApproachSynt

17、hesized peptidesPreview with human fetal liverEvaluation of technologiesMRPP,HPPP,PSI,HAI,etc.Why is liver?wwwww.HLPP.HUPO为何研究肝脏为何研究肝脏?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。药药 物物毒毒 物物营养物营养物

18、生物异源物生物异源物生物转化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 1.2107种以上的化学物种以上的化学物(CA Services list)以每周约以每周约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 大约大约 11,500种，作为食物或药物制剂添加物摄入种，作为食物或药物制剂添加物摄入 其余的其余的50,000种，为潜在的环境污染物种，为潜在的环境污染物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”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

19、（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 genom

20、ics Regenerome(再生组再生组)全球及中国肝炎的流行病学统计 全球全球:3.5:3.5亿携带者亿携带者中国中国:1.8:1.8亿携带者亿携带者死亡死亡:23:23万人万人/年年疾病负担疾病负担:500:500亿元人民币亿元人民币治疗手段治疗手段:抗病毒抗病毒,保肝降保肝降 酶酶,抗纤维化抗纤维化,免疫治疗等免疫治疗等缺点缺点:病毒易反弹病毒易反弹,病病 情易反复情易反复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HBV

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

22、性为15%15%全球全球:100:100万人万人/年年中国中国:50:50万人万人/年年死亡死亡:约约4545万人万人/年年疾病负担疾病负担:400:400亿元人民币亿元人民币治疗手段治疗手段:手术切除手术切除(只有只有15%-20%)15%-20%)治疗效果治疗效果:术后易转移术后易转移,易复易复发发,五 年 存 活 率五 年 存 活 率40-50%40-50%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原发性肝癌的治疗水平原发性肝癌的治疗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

23、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 这些病人术后这些病人术后5 5年生存率仅为年生存率仅为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

24、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肝脏是人类蛋白质组计划的首批目标！Prometheus stole fire from the gods and gave it to mortals.Prometheus BoundHeracles Libera

25、te Prometheus HLPPModern HLPPModern Prometheus MythPrometheus MythPrometheusPrometheus LiverLiver Eagle Liver diseases Eagle Liver diseases Heracles HLPPHeracles HLPP人类肝脏蛋白质组计划Human Liver Proteome Project(HLPP)国际国际HLPP共同体共同体Generate an integrative approach leading to a comprehensive functional map o

26、f 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 InitiativesVISIONHUPO Workshop,Bethesda,April 28-29,2002 Broad interests

27、and supports for initiating HLPP HUPO Liver Proteome Workshop,Beijing,October 22-24,2002 Getting consensus view for scientific objectives of HLPP HUPO 1st World Congress,Versailles,November 21-24,2002 Co-chairs of HLPP:Drs.Fuchu He,John Bergeron and Christian Brchot HLPP Planning Committee:17 member

28、s;May 2003Proposals about Working Plan of HLPP Dr.John Bergeron:Jan 31,2003(Management);Dr.Fuchu He:Feb 17,2003(Scientific Strategy)Dr.Fuchu He:Mar 22,2003(Action Plan);Dr.Christian Brechot:Mar 31,2003(Sampling)HLPP Office,March 18,2003 HUPO LPP Workshop,Bethesda,July 17-18,2003 NIH participatingHUP

29、O 2nd World Congress,Montreal,Oct.8-12,2003 Dr.Fuchu He was appointed as the executive Chair of HLPP(2003-2005)HUPO HLPP Satellite Meeting,Montreal,Oct.12,2003 Set up Executive Committee&9 Sub-committees,Action Plan at Pilot PhaseFrench liver samples were collected and distributed among 8 labs,May,2

30、004HUPO HLPP Satellite Meeting,Beijing,Oct.24,2004Chinese part of HLPP was officially launched,Nov.,2004Chinese liver samples were collected and distributed among 7 labs,Feb.,2005MISION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SLpSTSGESLYHVLGLDK)or othe

31、r 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

32、 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

33、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?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测基因证实推测基因对比调控模式对比调控模式 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”

34、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qSOPsqSpecimen BankingqPlatformsqWorking teamqGlobal CollaborationqExpression ProfileqORFeome qAntibodiesqBioinformatics qq Modification Profileq Subcellular Localizationq Protein-Protein Interactionq Antibodiesq Bioinformaticsq Integrate proteome with transcrip

35、tomeq Compare proteomes within health and diseased liverPilot Phase(2003-2005)Phase II(2006-2010)Outline BackgroundProgress of the HLPP-Progress report on expression profilingInitiation of the CNHLPPNext workProgress-Expression profilingEvaluation of technologiesBioinformatics set-upSampling and pre

36、parationPreview with human fetal liverPrimary analysis of French liver tissues ProgressEvaluation of technologiesBioinformatics set-upSampling and PreparationPreview with human fetal liverPrimary analysis of French liver tissues Major conclusions from the valuation of technologies 16 standard protei

37、ns/7 labsnHigh molecular weight and low abundance proteins are less detectable by 2DE.nProteins with only three order of magnitude could be identified in 2DE,but proteins with four order of magnitude could be identified in shotgun technology.nMore peptides(redundant)were identified for high MW prote

38、ins in shotgun technology.nSome similar proteins were identified(not protein group),suggesting the criteria for distinguishing similar proteins should be more strict.ProgressEvaluation of technologiesBioinformatics set-upSampling and PreparationPreview with human fetal liverPrimary analysis of Frenc

39、h liver tissues HLPP Experimental DatabaseMWS Data ClientGUI，Web Services,APIFiles Interchange,DMBS DumpData synchronizeData submissionExperiment reports,data,files HLPP Data MirrorMWS Data ClientMWS Data ClientMWS Data ClientMWS Data ClientMWS Server AdministratorHLPP Data MirrorSubmission Packages

40、 RepositoryHLPP Project Management DatabaseHLPP Project Management SystemHLPP Participating LaboratoriesHLPP Data CenterThe HLPP Data Management System Architecture Based on MyWorkSpace System(MWS)Current Standard draft of HLPPSample InformationProtein extractionConcentration MeasurementSeparationGe

41、l-based2DEGel1D-IEFGel1D-SDSLC-basedRPLCSCXLCSAXLCSECTrapDesaltingDigestionIon sourcenMALDIAutoflexUltraflexABI 4700nESIQstarQTofMicroLCQLTQMass spectrometrynTOFnTOFTOFUltraflexABI 4700nQTOFQstarQTofMicronITMSLCQLTQPeak list generationABI 4700QStarAutoflexTurboSequestMasslynxFlexAnalysisPeak list pr

42、eprocessingGPS peak list preprocessProtein/peptide identificationMascotSequestProtein list generationnAutoflexnGPSnBiotoolsnQstarnBuildSummarynDTASelectnBioworksnAutoQuest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 pathwaysM

43、yeloma 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

44、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 tissuesgroup(A,B)protein Aprotein Bdetected peptides Protein i

45、dentification 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.Definition of Protein GROUP Criteria for protein identific

46、ationnIon trap XCorr(highest)1.9(1),2.2(2),3.75(3);DeltaCn0.1;RSp4.nABI-TOF/TOF Protein score59(Rank 1 for the spot)nQ-TOF or Q-STAR Protein score threshold Peptide score “the score for identity”or 29 nMALDI-TOF Protein score threshold The proteins in first report(multi-protein for the mixture)Progr

47、ess Evaluation of technologies Bioinformatics set-up Sampling and preparation Preview with human fetal liver Primary analysis of French liver tissues Evaluation of the SOPs for the subcellular fractionation Object:Find the optimum method of pretreatment Sample:Liver tissues of C57 mice Subcellular:M

48、itochondria,Golgi,PM,Nucleus,ER,Cytoplasm Sample pretreatment Fresh tissues Frozen homogenised tissues Frozen tissuesPurity(Western blotting):Fresh tissues frozen homogenized tissues frozen tissuesThe yield of proteins:Fresh tissues frozen homogenized tissues frozen tissuesIntegrity(TEM):Fresh tissu

49、es frozen homogenized tissuesSummaryProgressEvaluation of technologiesBioinformatics set-upSampling and PreparationPreview with human fetal liverPrimary analysis of French liver tissues It is a giant ambitious objective and gives a great challenge to embryonic proteomics It is necessary to carry out

50、 a preview before the formal launching of this projectScience 302:1316,Nov.21,2003Nature 425:441,Oct.2,2003Average Throughput of the Platform 2DE System 12 Gel/3 Days Auto-Spot Digest System 1152 spots/Day MS and MS/MS System 500-1000 Spots/Day The area scales before and after isoform processing in