复旦大学：《分析化学》科学讲座_肿瘤蛋白质标志物研究的新技术新方法（陆豪杰）

肿瘤蛋白质标志物研究的新技术新方法 陆豪杰 复旦大学化学系&生物医学研究院

肿瘤蛋白质标志物研究的新技术新方法 陆豪杰 复旦大学化学系 &生物医学研究院

2002 John B Fenn, Koichi Tanaka, John B Fenn and Koichi Tanaka"for their development of soft Kurt Wuthrich desorption ionisation methods for mass spectrometric analyses of biological macromolecules"and Kurt Wuthrich"for his development of nuclear magnetic resonance spectroscopy for determining the three-dimensional structure of biological macromolecules in solution 2003 Peter Agre, Roderick to Peter Agre"for the discovery of water channels"and with one Mackinnon half to roderick mackinnon "for structural and mechanistic studies of ion channels" 2004 Aaron Ciechanover, Avram For the discovery of ubiquitin-mediated protein degradation Hershko, Irwin rose 2005 Yves Chauvin Robert H For the development of the metathesis method in organic synthesis Grubbs and richard r. schrock 2006 Roger D Kornberg For his studies of the molecular basis of eukaryotic transcription 2007 Gerhard Ertl For his studies of chemical processes on solid surfaces 2008 Osamu Shimomura, Martin For the discovery and development of the green fluorescent Chalfie, Roger Y. Tsien protein, GFP 2009 Venkatraman Ramakrishnan, For studies of the structure and function of the ribosome Thomas A Steitz, Ada e. 2010 Richard F. Heck, Ei-ichi Negishi, For palladium-catalyzed cross couplings in organic synthesis Akira Suzuki Dan shechtman For the discovery of quasicrystals 2012 Robert J Lefkowitz, Brian K. For studies of G-protein-coupled receptors Kobilka

2002 John B. Fenn, Koichi Tanaka, Kurt Wüthrich John B. Fenn and Koichi Tanaka "for their development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules" and Kurt Wüthrich "for his development of nuclear magnetic resonance spectroscopy for determining the three‐dimensional structure of biological macromolecules in solution". 2003 Peter Agre, Roderick MacKinnon to Peter Agre "for the discovery of water channels" and with one half to Roderick MacKinnon "for structural and mechanistic studies of ion channels". 2004 Aaron Ciechanover, Avram Hershko, Irwin Rose For the discovery of ubiquitin‐mediated protein degradation". 2005 Yves Chauvin, Robert H. Grubbs and Richard R. Schrock For the development of the metathesis method in organic synthesis 2006 Roger D. Kornberg For his studies of the molecular basis of eukaryotic transcription 2007 Gerhard Ertl For his studies of chemical processes on solid surfaces 2008 Osamu Shimomura, Martin Chalfie, Roger Y. Tsien For the discovery and development of the green fluorescent protein, GFP 2009 Venkatraman Ramakrishnan, Thomas A. Steitz, Ada E. Yonath For studies of the structure and function of the ribosome 2010 Richard F. Heck, Ei‐ichi Negishi, Akira Suzuki For palladium‐catalyzed cross couplings in organic synthesis 2011 Dan Shechtman For the discovery of quasicrystals 2012 Robert J. Lefkowitz, Brian K. Kobilka For studies of G‐protein‐coupled receptors

i(n /ll THE HUMAN D i1 1(:: 112n GENOME 78910112 上世纪90年代,科学家历经10年花费30亿美元完成人类第一张基因草图

上世纪90年代，科学家历经10年花费30亿美元完成人类第一张基因草图

Same genome Different Proteome 幼蟲期 成期 期 蝴蝶一生具有四个明显不同的发育阶段:(1)卵期(胚胎时期);(2)幼 虫期(生长时期);(3)蛹期(转变时期);(4)成虫期(有性时期)

Same Genome Different Proteome 蝴蝶一生具有四个明显不同的发育阶段：（1）卵期（胚胎时期）；（2）幼 虫期（生长时期）；（3）蛹期（转变时期）；（4）成虫期（有性时期）

蛋白质组学在转化医学中处于核心的地位 美国NH2007年公布:转换医学作为新世纪医学研究的主要目标 转化医学面临的挑战 转化医学的目标 生物标志物的革新 有效的诊断和预后 蛋白质组学 致病机理的系统描绘 >准确认药物靶标 准确评价疗效 活性和毒性的表征 病人间的个体差异 个性化医疗的应用 Zerhouni ea. director of nih 2007, publication in clinical pharmacology and therapeutics

蛋白质组学在转化医学中处于核心的地位 蛋白质组学 生物标志物的革新 致病机理的系统描绘 准确评价疗效 病人间的个体差异 转化医学面临的挑战 有效的诊断和预后 准确确认药物靶标 活性和毒性的表征 个性化医疗的应用 转化医学的目标 Zerhouni EA, Director of NIH, 2007, publication in clinical pharmacology and therapeutics 美国NIH 2007年公布:转换医学作为新世纪医学研究的主要目标

Background Categories of:Expression Structural Functional proteomics proteomics」 proteomics project Proteome Structure of Subcellular Biomarker Protein-protein The mechanisms or interactions functions of protein compartmentalization/ Proteomic data Genomie data RXR BRCA actin ras Integration Wnt Benefits Prove the existence of genes Elucidate the mechanisms of diseases, aging and protein functions Facilitate gene therapy, ete. 指应用各种技术手段来研究蛋白质组的一门新兴科学,其目 的是从整体的角度分析细胞内动态变化的蛋白质组成成份、 表达水亚与修饰状态,了解蛋白质之间的相互作用与联系, 蛋白质的定位,揭示蛋白质功能与细胞生命活动规律

Background Categories of proteomics Application Benefits • 指应用各种技术手段来研究蛋白质组的一门新兴科学，其目 的是从整体的角度分析细胞内动态变化的蛋白质组成成份、 表达水平与修饰状态，了解蛋白质之间的相互作用与联系， 蛋白质的定位，揭示蛋白质功能与细胞生命活动规律

2DE-MALDI MS based strategy Protein(s) Enz. digestion MS Analysis DB Search DB Identities Pep. mixture Experimental Experimental mass spectrum p Trypsin 2-D gel purification Theoretical ATCGCCGTA mass spectrum LMNVCWRTYPILHK TGACTAGGI GLKINHGTRFDSK RTYPILI MFGHDSAK ACCAGTAGA CAGTAGGTA HIDSAK CGTAGCAGT ATGCAGTAC Protein Theoretical DNA sequenc database peptides database M/Z

2DE-MALDI MS based strategy

LC-ESI based strategy Sample prep LC/MS Quantification Ms/Ms Abundances Pep. mixture DB search DB entities MS eparation Survey scan MS/MS scan peptides m/z Database searching L TSAGLGK Protein In silice database digest Theoretical spect

LC-ESI based strategy

蛋白质经胰蛋白酶酶切产生肽段的原理 Trypan c山an Peptide zaquanea RLVKEVALGYKIGRFGGKAGVRNTRV Digested frogments RLvk」! EVALGYK|工eR|Foek」 AGVRNTR ArgInine: R Lysine: K Trypsin cleavage

蛋白质经胰蛋白酶酶切产生肽段的原理

肽段碎裂产生碎片离子的原理 MS1 CID MS2 source = Fixed ∈ Scanning m/z H o H O DHLFR-sHK H-N-C. N-terminal fragments C-terminal fragments LIR-sHK H2N-C-CNH-CTC-NHFC-C-OH Rs-K X2y222x1y121 DLR 2'HaN-C-C-NH-C-C-OH D-LHRHs

肽段碎裂产生碎片离子的原理