A new method for analysis of disulfide-containing proteins by matrix-assisted laser desorption ionization (MALDI) mass spectrometry
A new method for analysis of disulfide-containing proteins by matrix-assisted laser desorption ionization (MALDI) mass spectrometry
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DOI：
10.1016/j.jasms.2009.08.020
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发表时间：
2009-12
影响因子：
3.2
通讯作者：
Hongmei Yang;Ning Liu;Xiaoyan Qiu;Shuying Liu
中科院分区：
文献类型：
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作者：
Hongmei Yang;Ning Liu;Xiaoyan Qiu;Shuying Liu
描述了一种利用肽-基质加合物鉴定含二硫键的肽的简单且高通量的方法。在MALDI质谱中，一些常用的基质可以特异性地与肽中的巯基发生反应，从而在二硫键连接的肽化学还原后的MALDI质谱中观察到肽-基质加合离子[M+n+ n ′基质+H]+或[M+n+n′基质+Na]+（n=半胱氨酸残基的数目，n′=1，2，.，n）。在测试的几种基质中，发现α-氰基-4-羟基肉桂酸（CHCA，分子量189 Da）和α-氰基-3-羟基肉桂酸（3-HCCA）对含二硫化物的肽/蛋白质的MALDI分析更有效。二硫键中涉及的两个还原半胱氨酸导致每个半胱氨酸的质量偏移为189 Da，因此可以确定二硫键的数量，而对于其他基质（芥子酸，阿魏酸和咖啡酸），除非在碱性条件下进行反应，否则不会发生类似的加成反应。提出了在巯基处的基质加成反应的基本机理，并研究了可能影响肽-基质加合物形成的几个因素。通常，该方法快速、有效且稳健地鉴定蛋白质/肽中的二硫键。
A simple and high-throughput method for the identification of disulfide-containing peptides utilizing peptide-matrix adducts is described. Some commonly used matrices in MALDI mass spectrometry were found to specifically react with sulfhydryl groups within peptide, thus allowing the observation of the peptide-matrix adduct ion [M+n+n′ matrix+H]+or [M+n+n′ matrix+Na]+(n= the number of cysteine residues,n′=1, 2,…,n) in MALDI mass spectra after chemical reduction of disulfide-linked peptides. Among several matrices tested, α-cyano-4-hydroxycinnamic acid (CHCA, molecular mass 189 Da) and α-cyano-3-hydroxycinnamic acid (3-HCCA) were found to be more effective for MALDI analysis of disulfide-containing peptides/proteins. Two reduced cysteines involved in a disulfide bridge resulted in a mass shift of 189 Da per cysteine, so the number of disulfide bonds could then be determined, while for the other matrices (sinapinic acid, ferulic acid, and caffeic acid), a similar addition reaction could not occur unless the reaction was carried out under alkaline conditions. The underlying mechanism of the reaction of the matrix addition at sulfhydryl groups is proposed, and several factors that might affect the formation of the peptide-matrix adducts were investigated. In general, this method is fast, effective, and robust to identify disulfide bonds in proteins/peptides.