Riviere L R, Tempst P
OsteoArthritis Sciences, Inc., Cambridge, Massachusetts, USA.
Curr Protoc Protein Sci. 2001 May;Chapter 11:Unit 11.1. doi: 10.1002/0471140864.ps1101s00.
Analysis of protein covalent structure is less complex and more accurate when performed on peptides derived from the larger protein. In contrast to acid-promoted total hydrolysis, peptides are typically generated by selective proteolysis, i.e., by specifically cleaving peptide bonds with endoproteases that have varying degrees of specificity. This unit presents a protocol that can be used to generate peptide fragments from intact, undenatured proteins. Fragments can be analyzed directly by mass spectrometry (MS) or, more often, are first separated by reversed-phase HPLC (RP-HPLC) and then analyzed by MS or automated sequencing. Most proteins are resistant to enzymatic proteolysis under nondenaturing conditions or are not soluble in aqueous solution. Digestion procedures performed in the presence of chaotropic agents and SDS are described, and support protocols provide instructions for preparing enzyme stocks and reducing and alkylating peptides prior to sequencing or HPLC analysis.
对源自较大蛋白质的肽进行蛋白质共价结构分析时，其过程不太复杂且更准确。与酸促进的完全水解不同，肽通常通过选择性蛋白水解产生，即通过使用具有不同程度特异性的内切蛋白酶特异性切割肽键来实现。本单元介绍了一种可用于从完整、未变性蛋白质生成肽片段的方案。片段可直接通过质谱（MS）分析，或者更常见的是，先通过反相高效液相色谱（RP-HPLC）分离，然后再通过MS或自动测序进行分析。大多数蛋白质在非变性条件下对酶促蛋白水解具有抗性，或者不溶于水溶液。文中描述了在离液剂和十二烷基硫酸钠存在下进行的消化程序，支持方案提供了制备酶储备液以及在测序或HPLC分析之前对肽进行还原和烷基化处理的说明。
