Butturini Elena, Boriero Diana, Carcereri de Prati Alessandra, Mariotto Sofia
Department of Neuroscience, Biomedicine and Movement Sciences, Section of Biological Chemistry, University of Verona, Verona, Italy.
MethodsX. 2019 Sep 10;6:1992-1998. doi: 10.1016/j.mex.2019.09.001. eCollection 2019.
S-glutathionylation is a reversible post-translational modification of proteins that generate a mixed disulfide between glutathione to thiolate anion of cysteine residues in target proteins. In the last ten years, S-glutathionylation has been extensively studied since it represents the cellular response to oxidative stress, in physiological as well as pathological conditions. This modification may be a protective mechanism from irreversible oxidative damage and, on the other hand, may modulate protein folding and function. Due to the importance of S-glutathionylation in cellular redox signaling, various methods have been developed to identify S-gluthationylated proteins. Herein, we describe two easy methods to recognized S-glutathionylation of a target protein after oxidative stress in cellular extracts based on different immunoprecipitation procedures. The immunoprecipitation assay allows the capture of one glutathionylated protein using a specific antibody that binds to the target protein. The presence of S-glutathionylation in the immunoprecipitated protein is identified using anti-glutathione antibody. The second type of approach is based on the detection of the glutathionylated protein with biotin/streptavidin technique. After different steps of protection of non-oxidized thiolic groups and reduction of S-glutathionylated groups, the newly-formed protein free-thiols are labeled with biotin-GSH. The modified protein can be isolate with streptavidin-beads and recognized using an antibody against target protein. •S-glutathionylation is a reversible post-translational modification of proteins that recently has been emerged as important signaling in the redox regulation of protein function.•Both methods to identify glutathionylated proteins are economic, easy and do not require particular equipment.•The setups of both methods guarantee high reproducibility.
S-谷胱甘肽化是一种蛋白质的可逆翻译后修饰，它在目标蛋白的半胱氨酸残基的硫醇阴离子与谷胱甘肽之间生成混合二硫键。在过去十年中，S-谷胱甘肽化受到了广泛研究，因为它代表了生理和病理条件下细胞对氧化应激的反应。这种修饰可能是一种防止不可逆氧化损伤的保护机制，另一方面，可能会调节蛋白质的折叠和功能。由于S-谷胱甘肽化在细胞氧化还原信号传导中的重要性，已经开发了各种方法来鉴定S-谷胱甘肽化的蛋白质。在此，我们描述了两种基于不同免疫沉淀程序的简便方法，用于识别细胞提取物在氧化应激后目标蛋白的S-谷胱甘肽化。免疫沉淀测定允许使用与目标蛋白结合的特异性抗体捕获一种谷胱甘肽化的蛋白质。使用抗谷胱甘肽抗体鉴定免疫沉淀蛋白中S-谷胱甘肽化的存在。第二种方法基于生物素/链霉亲和素技术检测谷胱甘肽化的蛋白质。在对未氧化的硫醇基团进行不同步骤的保护和S-谷胱甘肽化基团的还原之后，新形成的蛋白质游离硫醇用生物素-GSH进行标记。修饰后的蛋白质可以用链霉亲和素磁珠分离，并用针对目标蛋白的抗体进行识别。
•S-谷胱甘肽化是一种蛋白质的可逆翻译后修饰，最近已成为蛋白质功能氧化还原调节中的重要信号。
•两种鉴定谷胱甘肽化蛋白质的方法都经济、简便，且不需要特殊设备。
•两种方法的设置都保证了高重现性。
