DEVELOPMENT OF NEW METHOD FOR ISOLATING C TERMINAL PROTEOLYTIC PEPTIDE

C-末端肽的分离和MS表征 proteins. 蛋白质C末端的表征是 在识别和确认翻译后加工中很重要。 因此，它在质量控制中至关重要。 的一个主要 目前可用的方法的局限性是它的敏感性 （通常需要100-1000皮摩尔的样品）。 在这个项目中 我们开发了一种既快速又灵敏的新方法（3）。 的 该策略包括四个步骤：1）蛋白质降解 2）将C-末端肽与所有其它肽分离， 通过使用脱水胰蛋白酶（一种修饰的 胰蛋白酶，其仅结合在C末端含有K或R的肽） 3)C-末端肽的质量通过MALDI测定。 通过MS-MS获得C-末端肽的序列。 程序进行了测试，并显示出良好的工作对几种蛋白质高达 50 KDa。 然而，在排序方面没有实际限制 大蛋白质 整个过程大约需要30分钟， 蛋白质和几个样品可以并行分析， 三步 蛋白质含量低至10皮摩尔 成功测序。

Isolation & MS characterization of the c-terminal peptide of proteins. Characterization of the C-terminus of a protein is important in identifying and confirming post-translational processing. Thus it becomes essential in quality control. One of the major limitations of the methodology available today is its sensitivity (usually 100-1000 picomoles of sample are required). In this project we develop a novel procedure that is both fast and sensitive (3). The strategy involves four steps: 1) The protein is degraded enzymatically 2) The c-terminal peptide is separated from all other proteolysis products through the use of anhydrotrypsin (a modified trypsin that binds only peptides containing K or R at the C-terminus) 3) The mass of the c-terminal peptide is determined by MALDI 4) The sequence of the c-terminal peptide is obtained by MS-MS. The procedure was tested and shown to work well on several proteins up to 50 KDa. However, there is no practical limitation in sequencing bigger proteins. The whole procedure takes about 30 minutes for each protein and several samples can be analyzed in parallel for the first three steps. Proteins present at the level as low as 10 picomoles were successfully sequenced.