《蛋白质组学》PPT课件

1、,生命现象的物质基础是核酸和蛋白质，作为基因的载 体，核酸编制了所有的信息，而基因的表达产物蛋白 质，才是各种生物功能的执行者。,蛋白质组是功能基因组的新前沿，对基因表达的时空规律进行揭示，比较了不同生理条件下或不同发育时期的功能蛋白质组的差别和多样性（mRNA剪切的结果或翻译后加工的结果），才能在分子水平上全面、具体了解生命活动的过程，为个种生物学特征和生产应用提供理论基础。,基因,疾病,环境,REAL COMPLEXITY IS IN CELLULAR ROTEOMES,BEYOND THE GENOME Tissue Specific Expression Alternate Splic

2、ing, (1/3 of all genes) Post-Translational Modifications Types and Level: Signal Sequence cleavage Glycosylation Phosphorylation Farnylation Isoprenylation Acetylation All combine 100-1000 fold increase in complexity Future (challenging) research focus,1, 蛋白质组的基本概念和历史 2，蛋白质组的特征 3，蛋白质组与基因组的关系 4，蛋白质组的

3、研究技术和研究策略 5，蛋白质组的研究现状（国际和国内） 6，蛋白质组研究的范例,后基因组时代的蛋白质组学,1, 蛋白质组的基本概念和历史,蛋白质组定义,1，基因组表达的全部蛋白质。 2，在一种细胞/组织内存在的全部蛋白质。,Proteome,1994 M.Wilkins and K.W.Williams Macquarie University in Sydney Total Proteins Complement of a Genome,PROTEOME : PROTEins expressed by a genOME,Proteomics Large-scale Study of Pro

4、teins,Why is proteomics necessary,Displaying and studying the products of genes directly is an attractive way of studying disease and complex problem in biology,The study of gene expression can also be attempted at the level of mRNA (microarray.) However ,Why is proteomics necessary,You can have a p

5、rotein in the cell when its mRNA is no longer present; You can have lots of mRNA without tranlation of the message to protein,No good correlation between mRNA abudence and protein amout in a cell at a given time.,Why is proteomics necessary,Protein post-translational modification,Proteomics directly

6、 contributes to Annotation of genome,Protein subcellular location,The Proteome and Technology,The technology required to separate large number of proteins, to identify them, and to study their modifications are not straightforward,There are many more proteins in a proteome than genes in a genome, es

7、pecially for eukaryotes. various ways a gene is spliced in constructing mRNA various ways a protein can be post-translationally altered,蛋白质组学 结构蛋白质组学 功能蛋白质组学 亚细胞蛋白质组学 修饰蛋白质组学 相互作用组学,* protein micro-characterization for large-scale identification of proteins and their post-translational modifications

8、 * differential display proteomics for comparison of protein levels with potential application in a wide range of diseases * studies of proteinprotein interactions using techniques such as mass spectrometry or the yeast two-hybrid system,Proteomics,The proteome, unlike the genome, is not a fixed fea

9、ture of an organism. Instead, it changes with the state of development, the tissue or even the environmental conditions under which an organism finds itself.,Functional Proteomics,During human development, cell express different proteins Normal and cancer cells express different proteins Cell treate

10、d with and without drug express different proteins Post-translational, and covalent and non-covalent associations etc,蛋白质组研究简况,Initiation stage (1994-1997): - Term “proteome” was created by Wilkins, M. and Williams, K. in1994. - The first meeting on proteome was hold in 1997.,Progression stage (1998

11、-present): - Projects of proteomics were activated internationally. - Database of proteome were expanded. The milestone of proteomic research. Human Proteomics Organization (HUPO),Pre-stage (1970s-1994) - Two-dimensional electrophoesis - database - Human Protein Index,HUPO (The Human Proteome Organi

12、zation),Proteomics is in essence the study of the function, regulation and expression of proteins in relation to the normal function of the cell and in the initiation or progression of a disease state. Proteomics is of particular importance as it is the level of protein activity that most disease ar

13、e manifested. Consequently proteomics seeks to correlate directly the involvement of specific proteins and/or protein complexes in given disease state,2，蛋白质组的特征,蛋白质组的确定？,1，蛋白质组的完整性： 基因组内基因（或ORF）数量？ 基因表达？ 2，蛋白质组的测定： 修饰蛋白质的判据？ 蛋白质的动态变化（降解或转运）？,相互作用与蛋白质组研究,没有孤立的蛋白质 蛋白质相互作用的特点： 1）蛋白质网络 2）蛋白质复合体,蛋白质的“四维”

14、研究,特定的时间：合成与降解 特定的空间：区域性与运动性,蛋白质组研究技术的复杂性,20种化学和物理性质不同的氨基酸 蛋白质量的动态差别（106 ） 蛋白质的不稳定性 蛋白质修饰的多样性和复杂性,3，蛋白质组与基因组的关系,互相补充,转录水平 基因芯片,翻译水平 蛋白质组研究,互相帮助,1，基因组的注释（Annotation） 2, 蛋白质的质谱鉴定 3，依赖基因组研究技术的蛋白质组研究技术（双杂交技术、噬菌体显示技术 ）,结构基因组,功能基因组,结构蛋白质组,功能蛋白质组,蛋白质组研究不同于基因组研究,相对独立,紧密连接,4，蛋白质组的研究技术和研究策略,动物模型/细胞,蛋白质组研究,生物信

15、息学分析,功能分析,人类疾病,蛋白质组新技术,蛋白质组研究的策略：平台与整合,蛋白质组生物信息学,Bench works (2D, MS, etc),Databases,Bioinformatics,Questions,1. Wide Dynamic Range 2. Various Post-translation Modification Huge Amount of Species Four-dimensional Behaviour (3D Location, Timing),Solutions Different identification and separation techn

16、iques connected to meet different needs Alternative Selections,Solutions Combination of instrumentation and protein chemistry,Solutions Robust, Automation Proteomic techniques combined with bioinformatics for high-throughput data processing,Solutions Fractionation, subcellular proteomics Reproducibl

17、e control of sample preparation,Proteome Research,Cell Proteins Separation and Resolution Identification Structure and Function of proteins,蛋白质组学的主要研究方法,1、基于二维电泳-质谱技术的蛋白质组研究,2、基于质谱技术的蛋白质组研究 液相电泳-质谱技术 液相色谱-质联用技术 Shotgun质谱技术 ICAT技术,3、蛋白质芯片,High Vacuum System,Inlet,Ion Source,Mass Filter,Detector,Data

18、System,Sample Target HPLC GC CE,MALDI ESI Ion Spray API LSIMSS EI CI,TOF Quadruple Ion Trap Magnetic Sector FTMS,Microchannle Electron Multiplier Hybrid,PC SunSPAR,Mass Spectrometry,Mass Spectrometry,Generalized schematic of proton energy absorption by the matrix in MALDI.,TOF-MS instrument with a s

19、ingle acceleration stage,Principle of PSD analysis in MALDI/MS,FEq (1) F = ma (2) a = Eq/m (3),E S I Electrospray Source,Triple-quadrupole ESI mass spectrometer possesses ion selection and fragmentation capabilities.,ESI-ion trap,LC-ESI-MS,Micro-column (2.1mm-1.0mm) + Normal Spray pmole Capillary co

20、lumn(1.0mm-0.1mm) + Microspray fmole Nanoflow colmn ( 0.1mm) + Nanospray amole,Liquid Chromatography Electrospray Ionization Mass Spectrometry,Designation for peptide CID fragment ions,NH2-CH C NH CH-C-NH-CH-C-OH,O,R1,R2,R3,O,O,an,bn,cn,xn,yn,zn,H+,Comparison of ESI and MALDI,ESI MALDI,Charge multip

21、le single Sample preparation liquid solid Speed slow rapid capacity small large MS/MS capability strong weak,MS for Characterization of Proteins and peptide,Mass Determination Peptide mass fingerprinting,Database identification,Sequence-specific MS/MS,MS for Characterization of Proteins and peptide,

22、Peptide mass fingerprinting (MALDI-TOF),Database theoretical digestion,Digestion,Matching & Scoring,Digestion,MS for Characterization of Proteins and peptide,Peptide Mass,Database identification,Sequence-specific MS/MS,GHVASTYK,Digestion,SEQUESTTM How It Works,SEQ 1 SEQ 2 SEQ 3 SEQ 4,ESI/LC/MSn SEPA

23、RATION,MS plus MS/MS FRAGMENTATION,USER-GENERATED MS/MS SPECTRUM,STEP 1.,Why Mass Spectrometry for Proteomics?,Protein/Peptide Mass Accuracy Sequence Information Sensitivity Speed Direct Analysis of Protein Mixtures Post-translational Information Phosphorylation Glycosylation Quantitation (Expressio

24、n),New MS Techniques,MALDI-ion trap ESI-QqTOF MALDI-QqTOF ESI-Q-ion trap .,ESI/MALDI- Qq-TOF,Two dimensional electrophoresis,1975,High-resolution IEF-SDS-PAGE,O Farrell, Klose Scheele,Separation of over 1000 proteins in E. coli,Drawback: carrier ampholytes used to generate pH gradients is difficult

25、to achieve good reproducibility. Limited loading amount.,Two-dimensional Electrophoresis (2-DE),1st dimension: isoelectric focusing (IEF) proteins are separated in a pH gradient ubtil reach a stationary position where their net charge is zero. The pH at which a protein has zero net charge is called

26、isoelectric point (pI),2nd dimension: SDS-PAGE,Proteins are separated according thire molecular weight (Mr),Two-dimensional Electrophoresis (2-DE),!980s The introduction of immobilised pH gradient (IPG) eliminlated the problem of gradient instability and poor sample loading capacity,Two-dimensional

27、Electrophoresis (2-DE),Immobilised pH gradient (IPG) pH gradient is co-polymerised with the acrylamide gel matrix to form completely stable gradients. The commercial precast IPG stripes is available in various pH ranges (3-!0,4-7.).,Two-dimensional Electrophoresis (2-DE),2-DE is the only methode currently available which is capable of simultaneously separating thousands of proteins,Two-dimensional Electrophoresis (2-DE),!980s The introduction of immobilised pH gradient (IPG) eliminlated the problem of gradient instability and poor sample loading capacity,Sampl