Grant Gregory A
Washington University School of Medicine, Department of Medicine and Department of Developmental Biology, St. Louis, Missouri.
Curr Protoc Protein Sci. 2017 Feb 2;87:15.1.1-15.1.23. doi: 10.1002/cpps.22.
This unit describes a number of methods for modifying cysteine residues of proteins and peptides. A general procedure for alkylation of cysteine residues in a protein of known size and composition with haloacyl reagents or N-ethylmaleimide (NEM) is presented, and alternate protocols describe similar procedures for use when the size and composition are not known and when only very small amounts of protein are available. Alkylations that introduce amino groups using bromopropylamine and N-(iodoethyl)-trifluoroacetamide are also presented. Two procedures that are often used for subsequent sequence analysis of the protein, alkylation with 4-vinylpyridine and acrylamide, are described, and a specialized procedure for 4-vinylpyridine alkylation of protein that has been adsorbed onto a sequencing membrane is also presented. Reversible modification of cysteine residues by way of sulfitolysis is described, and a protocol for oxidation with performic acid for amino acid compositional analysis is also provided. Gentle oxidation of cysteine residues to disulfides by exposure to air is described. Support protocols are included for recrystallization of iodoacetic acid, colorimetric detection of free sulfhydryls, and desalting of modified samples. © 2017 by John Wiley & Sons, Inc.
本单元介绍了多种修饰蛋白质和肽中半胱氨酸残基的方法。文中给出了一种使用卤代酰试剂或N - 乙基马来酰亚胺（NEM）对已知大小和组成的蛋白质中的半胱氨酸残基进行烷基化的通用程序，以及替代方案，描述了在大小和组成未知以及仅获得极少量蛋白质时使用的类似程序。还介绍了使用溴丙胺和N - （碘乙基） - 三氟乙酰胺引入氨基的烷基化反应。描述了两种常用于蛋白质后续序列分析的程序，即与4 - 乙烯基吡啶和丙烯酰胺的烷基化反应，还给出了一种用于已吸附在测序膜上的蛋白质的4 - 乙烯基吡啶烷基化的专门程序。描述了通过亚硫酸解对半胱氨酸残基进行可逆修饰的方法，还提供了一种用过甲酸氧化进行氨基酸组成分析的方案。介绍了通过暴露于空气中将半胱氨酸残基温和氧化为二硫键的方法。包括碘乙酸重结晶、游离巯基的比色检测以及修饰样品脱盐的支持性方案。© 2017约翰威立父子公司。
