蛋白质组学Sample Prep.pptVIP

PROTEOMICSProtein Sample Preparation and Electrophoresis Ubiquitation IS THE CENTRAL DOGMA STILL VALID? One gene normally codes for more than one gene product (protein); The different proteins coded by the same gene have very often rather different functions; The different proteins coded by the same gene participate very often in different active complexes; Post-translational modifications of the gene products normally determine the activity of the gene. IS THE CENTRAL DOGMA STILL VALID? 一个基因不能直接决定一个蛋白质的功能； 人类大部分疾病不是基因改变造成的； 蛋白质也存在类似于mRNA分子内的剪切、拼接，这种自主性不能从其基因编码序列中预测； 基因是遗传信息的源头，而功能性蛋白是基因功能的执行体。基因组计划的实现为生物有机体全体基因序列的确定、为未来生命科学研究奠定了坚实的基础，但是它并不能提供认识各种生命活动直接的分子基础，其间必须研究生命活动的执行体-蛋白质； 蛋白质组学、蛋白质及其相互作用、蛋白质结构与功能研究将是未来生命科学研究的中心。 ProteomicsFrom One Revolution to the Next Welcome to ProteomicsThe Role of Post-translational Modifications Key Technologies in Proteomics Fractionation, 2D, MS, Bioinformatics Why Proteomics Have Been Developed Proteomics relies on integration of the significant advances recently achieved in: Separation technique (resolution and reproducibility) Mass spectrometry (MALDI-TOF and MS/MS) Bioinformatics (database sequence comparison) Sample Preparation and Separation 1. Introduction General differences between protein and DNA/RNA Technical differences between proteomics and genomics 2. Why needs to be prepared and separated ? 3. What preparation and separation techniques used in proteomics? Selective separation of a group of proteins ? Separation of all proteins in a cell or tissue ? 4. Which strategy is the best one? Protein Sample Preparation To completely extract the total proteins To remove non-proteins Factors should be considered 1. Is the sample from cells or solid tissue? 2. Is pre-fractionation desired? 3. What kind of interfering substances are present? 4. Quality of separation vs. total protein representation? Protein Extraction 1. Cell or Tissue Sample Disruption 2. Protein Precipitation 3. Protein Solubilizati