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.

描述了一种利用肽基质加合物鉴定含二硫化物肽的简单且高通量的方法。发现 MALDI 质谱中一些常用的基质与肽内的巯基发生特异性反应，从而可以观察肽-基质加合离子 [M+ n + n ' matrix+H] ⁺或 [M+ n + n ' matrix+ Na] ⁺ ( n = 半胱氨酸残基数，n '=1, 2,..., n) 在二硫键连接肽的化学还原后的 MALDI 质谱中。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）对含二硫化物肽的 MALDI 分析更有效/蛋白质。参与二硫键的两个还原半胱氨酸导致每个半胱氨酸的质量移动 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），类似的添加除非反应在碱性条件下进行，否则反应不会发生。提出了在巯基上的基质加成反应的潜在机制，并研究了可能影响肽-基质加合物形成的几个因素。