Gould Neal S
Department of Pediatrics, Children's Hospital of Philadelphia Research Institute, Philadelphia, PA, USA.
Methods Mol Biol. 2019;1967:183-195. doi: 10.1007/978-1-4939-9187-7_11.
The wide reactivity of the thiol group enables the formation of a number of chemically and biologically distinct posttranslational modifications. Proteins within nearly all major families undergo some form of cysteine modification and the modifications are associated with regulatory functions across many biological processes. However, the susceptibility of thiols to redox shifts, as well as the labile nature of most thiol modifications, renders detection difficult. Analysis difficulties are compounded further in complex protein mixtures due to the typical low abundance of cysteine modifications under normal physiological conditions. Here we describe methods for the analysis of three cysteine modifications: nitrosylation, glutathionylation, and S-acylation. The three methods use the same organic mercury-conjugated agarose resin as an enrichment platform. To date, over 2154 sites on 1446 proteins have been identified between the three modifications using this method. Using equivalent processing, enrichment, and analytical methods has enabled a more comprehensive picture of the redox proteome landscape.
硫醇基团的广泛反应性使得能够形成许多化学和生物学上不同的翻译后修饰。几乎所有主要蛋白质家族中的蛋白质都会经历某种形式的半胱氨酸修饰，并且这些修饰与许多生物过程中的调节功能相关。然而，硫醇对氧化还原变化的敏感性以及大多数硫醇修饰的不稳定性质使得检测变得困难。由于在正常生理条件下半胱氨酸修饰的典型低丰度，在复杂蛋白质混合物中的分析困难进一步加剧。在这里，我们描述了三种半胱氨酸修饰的分析方法：亚硝基化、谷胱甘肽化和S-酰化。这三种方法使用相同的有机汞偶联琼脂糖树脂作为富集平台。迄今为止，使用这种方法在三种修饰之间已鉴定出1446种蛋白质上的2154多个位点。使用等效的处理、富集和分析方法能够更全面地了解氧化还原蛋白质组概况。
