Morris H R, Pucci P, Panico M, Marino G
Department of Biochemistry, Imperial College of Science, Technology and Medicine, University of London, U.K.
Biochem J. 1990 Jun 15;268(3):803-6. doi: 10.1042/bj2680803.
In this paper we present a protocol that allows a dynamic analysis of disulphide-bridge formation, based on freezing the intermediates by acid/acetone precipitation, followed by digestion with pepsin and direct fast-atom-bombardment mass-spectrometric analysis. A rapid definition of the exact nature of disulphide bridges formed can be obtained via a definitive assignment of disulphide-linked peptides according to their unique mass values. With the use of an appropriate thiol concentration, scrambling of the native disulphide bonds in bovine insulin occurs, and the process is catalysed by protein disulphide-isomerase (EC 5.3.4.1). The disruption of native and the formation of new disulphide bonds can be monitored as described above, and interestingly B-chain dimers containing Cys-B7-Cys-B7 and Cys-B7-Cys-B19 bonds are detected.
在本文中，我们提出了一种方案，该方案基于通过酸/丙酮沉淀冷冻中间体，随后用胃蛋白酶消化并进行直接快速原子轰击质谱分析，从而允许对二硫键形成进行动态分析。通过根据二硫键连接的肽段独特质量值进行明确归属，可以快速确定所形成二硫键的确切性质。使用适当的硫醇浓度时，牛胰岛素中的天然二硫键会发生重排，并且该过程由蛋白质二硫键异构酶（EC 5.3.4.1）催化。天然二硫键的破坏和新二硫键的形成可以如上所述进行监测，有趣的是，检测到了含有Cys-B7-Cys-B7和Cys-B7-Cys-B19键的B链二聚体。
