Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
1] Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA. [2] Genentech Inc., South San Francisco, California, USA.
Nat Protoc. 2014 Jan;9(1):64-75. doi: 10.1038/nprot.2013.161. Epub 2013 Dec 12.
Reversible modifications of cysteine thiols have a key role in redox signaling and regulation. A number of reversible redox modifications, including disulfide formation, S-nitrosylation (SNO) and S-glutathionylation (SSG), have been recognized for their significance in various physiological and pathological processes. Here we describe a procedure for the enrichment of peptides containing reversible cysteine modifications. Starting with tissue or cell lysate samples, all of the unmodified free thiols are blocked using N-ethylmaleimide (NEM). This is followed by the selective reduction of those cysteines bearing the reversible modification(s) of interest. The reduction is achieved by using different reducing reagents that react specifically with each type of cysteine modification (e.g., ascorbate for SNO). This protocol serves as a general approach for enrichment of thiol-containing proteins or peptides derived from reversibly modified proteins. The approach uses a commercially available thiol-affinity resin (thiopropyl Sepharose 6B) to directly capture free thiol-containing proteins through a disulfide exchange reaction, followed by on-resin protein digestion and multiplexed isobaric labeling to facilitate liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based quantitative site-specific analysis of cysteine-based reversible modifications. The overall approach requires a simpler workflow with increased specificity compared with the commonly used biotinylation-based assays. The procedure for selective enrichment and analyses of SNO and the level of total reversible cysteine modifications (or total oxidation) is presented to demonstrate the utility of this general strategy. The entire protocol requires ∼3 d for sample processing with an additional day for LC-MS/MS and data analysis.
半胱氨酸巯基的可逆修饰在氧化还原信号转导和调控中起着关键作用。许多可逆氧化还原修饰，包括二硫键形成、S-亚硝基化（SNO）和 S-谷胱甘肽化（SSG），已被认为在各种生理和病理过程中具有重要意义。本文描述了一种富集含有可逆半胱氨酸修饰的肽的方法。从组织或细胞裂解物样品开始，使用 N-乙基马来酰亚胺（NEM）封闭所有未修饰的游离巯基。然后，使用不同的还原试剂选择性还原感兴趣的具有可逆修饰的半胱氨酸。还原反应是通过使用与每种类型的半胱氨酸修饰特异性反应的还原试剂（例如，抗坏血酸用于 SNO）来实现的。该方案是一种从可逆修饰的蛋白质衍生的含硫醇的蛋白质或肽的富集的通用方法。该方法使用市售的巯基亲和树脂（巯基丙基 Sepharose 6B）通过二硫键交换反应直接捕获含游离巯基的蛋白质，然后在树脂上进行蛋白质消化和多重等压标记，以促进基于液相色谱-串联质谱（LC-MS/MS）的半胱氨酸基可逆修饰的定量位点特异性分析。与常用的基于生物素化的测定相比，该方法具有更简单的工作流程和更高的特异性。选择性富集和分析 SNO 以及总可逆半胱氨酸修饰水平（或总氧化）的过程被提出以证明这种通用策略的实用性。整个方案需要大约 3 天的样品处理时间，外加一天的 LC-MS/MS 和数据分析时间。
