《蛋白质组学》.ppt

* * * PVM,并行计算机 MPI，并行编程技术 * * * * * * * * FCS Foetal Calf Serum * * * SDSLC-MS/MS In-Gel trypsin digestion LC-MS/MS PPT纲要 蛋白质鉴定 比较蛋白质组学 质谱鉴定蛋白质的原理及具体流程 双向凝胶电泳 初分离技术--SDS, antibody column, PF2D LC-MS/MS 2DE-MALDI-TOF/TOF 双向差异凝胶电泳(DIGE) 同位素标记技术 数据分析软件 (Sequest, X!tandem, TPP) 数据验证 无标记定量技术 Multi-dimensional Chromatography strong cation exchange (SCX) and reversed phase chromatography (RP ). Summary of Multi-Dimensional Chromatography Strengths: ? Reduces sample complexity ? Low abundance proteins ? Membrane proteins ? All MW Proteins ? Flexible (different column combinations) ? Can automate Limitations: ? Complex setup ? No faster than 2D gels ? Computer intensive ? Not Quantitative ? Reproducibility Mass Spectrometry BasedTechniqueswithStable Isotopes or MassTags Absolute Quantification (AQUA) Scheme 18O-labeling Protocol Quantify by 18O to 16O peptide ratios Strengths/Limitations of 18O-Labeling Strengths: ? No extra steps ? Relative quantification using multiple peptides from same protein ? Post-translational modification information ? 18O labeling kit (Prolytica) Limitations: ? No reduction in sample complexity ? Only 4 amu mass increase ? 18O exchange with water ? Quantification error ~20% Metabolic isotope labeling Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) developed by Matthias Mann in 2002 Quantify by 13C to 12C-Arg-labeled peptide ratios SILACStrengths vs. Limitations Strengths: incorporating efficiency nearly 100% does not require multiple chemical processing and purification steps Labeling essential amino acids such as arginine, lysine, leucine Limitations: Limit to active cell line and lower organism (yeast, bacterial, worm) 数据解析 蛋白质组数据的产出量和复杂性； 贝叶斯统计学解决基于肽质量指纹图谱鉴定蛋白质； 蛋白质组学杂志发布蛋白质鉴定准则，据此规范发表的数据； 发现蛋白质修饰的软件以被用于肽质量和肽段碎片数据分析； 糖链结构数据库和糖链质量指纹图谱的结构归属工具分析蛋白质糖基化。 * 泛素（英语:Ubiquitin）是一种存在于大多数真核细胞中的小蛋白。它的主要功能是标记需要分解掉的蛋白质，使其被水解。 蛋白质的糖基化是指在糖基转移酶作用下将糖转移至蛋白质，和蛋白质上的氨基酸残基形成糖苷键的过程。糖基化是对蛋白的重要的修饰作用，有调节蛋白质功能作用。 磷酸化