第五章蛋白结构分析技术.pptVIP

第五章 结构分析 蛋白质的氨基酸序列分析 生物质谱 X-射线晶体衍射技术 核磁共振 溶液中蛋白质分子构象的研究方法 蛋白质组 第一节 蛋白质和多肽的 氨基酸序列分析 Synopsis: 1. A practical approach to determining amino acid sequence requires a method to remove one amino acid at a time from a polypeptide chain. 2. This is achieved by the use of phenylisothiocyanate（异 硫氰酸苯酯）as the N-terminal labelling reagent, the Edman method. 3. This technique can only deal with chains of 20 to 60 amino acids, and most proteins are much larger. 4. To complete the process, selective hydrolysis of the polypeptide chain cuts very long polypeptides into specific fragments of more manageable size. N端序列分析 Sanger was the first person to determine a protein sequence, for insulin in 1953. The problem: after labelling the N-terminal amino acid, the polypeptide chain must be hydrolysed to isolate and identify the labelled amino acid. Whats needed is a way to remove the first amino acid without having to hydrolyze the whole chain. This was solved by Per Edman, in Sweden in 1956, by using the reagent phenylisothiocyanate to label the N-terminal end of the polypeptide sample. It works by cleaving the N-terminal AA off and leaving the rest of the peptide intact. The Edman method can be repeated on the peptide remaining after the reaction, so this is a very effective method for obtaining AA sequence information. In fact the whole sequence of a peptide containing many AAs can be determined by Edman method. 降解反应分三步 偶联 PITC的异硫氰酸酯基与蛋白的自由?－氨基作用，产生苯氨基硫甲酰蛋白质。 裂解 PTC－蛋白质在无水条件下酸裂解，切下反应的那个残基，暴露出下一个自由?－氨基。 转化 ATZ－氨基酸不稳定，在三氟乙酸水溶液条件下，转化成稳定的PTH－氨基酸。 PTH－氨基酸的鉴定 薄层层析，HPLC, GC(气相色谱），质谱分析等。 蛋白质N端测序仪器分析法： 1. 液相旋转杯序列仪 最早，很少用 2. 固相序列分析仪 有些氨基酸不能正确测定 3. 气相顺序仪 灵敏度很高，降低费用 4. 新型蛋白测序仪 快速可靠，高自动化 二. C端序列分析 适用 1. 鉴定重组蛋白是否正确表达 2. 大规模生产时质量控制 3. N端封闭蛋白的分析 4. DNA探针的设计 原理 化学试剂与蛋白质和多肽的羧基反应， 反应后的C末端衍生物被切割下来，通 过HPLC系统进行分析。 步骤 活化 酰胺化保护侧链羧基 烷基化 修饰Thr和Ser的羟基 裂解和衍生 第二节 生物质谱技术 Mass Spectrometry (MS) 是带电原子，分子 或分子碎片按质荷比(m/z