Resin-assisted enrichment of thiols as a general strategy for proteomic profiling of cysteine-based reversible modifications.
Resin-assisted enrichment of thiols as a general strategy for proteomic profiling of cysteine-based reversible modifications.
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DOI：
10.1038/nprot.2013.161
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发表时间：
2014-01
期刊：
影响因子：
14.8
通讯作者：
中科院分区：
文献类型：
--
作者：
半胱氨酸巯基的可逆修饰在氧化还原信号传导和调节中起重要作用。许多可逆的氧化还原修饰，包括二硫键形成，S-亚硝基化和S-谷胱甘肽化，已被公认为在各种生理和病理过程中的重要性。在这里，我们描述了一个程序的肽含有可逆的半胱氨酸修饰的富集。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硫醇。随后选择性还原那些带有感兴趣的可逆修饰的半胱氨酸。通过使用与每种类型的半胱氨酸修饰特异性反应的不同还原试剂（例如，抗坏血酸盐用于S-亚硝基化）。该方案用作富集含巯基蛋白质或衍生自可逆修饰蛋白质的肽的一般方法。该方法利用市售的巯基亲和树脂（硫丙基琼脂糖6 B），通过二硫键交换反应直接捕获游离的含巯基蛋白质，然后进行树脂上蛋白质消化和多重同量异序标记，以促进基于LC-MS/MS的半胱氨酸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，整体方法需要更简单的工作流程和更高的特异性。选择性富集和分析S-亚硝基化和总可逆半胱氨酸修饰（或总氧化）的水平的程序，以证明这种一般策略的效用。整个方案需要约3天的样品处理时间，另外一天用于LC-MS/MS和数据分析。
Reversible modifications of cysteine thiols play a significant role in redox signaling and regulation. A number of reversible redox modifications, including disulfide formation, S-nitrosylation, and S-glutathionylation, 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 S-nitrosylation). This protocol serves as a general approach for enrichment of thiol-containing proteins or peptides derived from reversibly modified proteins. The approach utilizes 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 LC–MS/MS based quantitative site-specific analysis of cysteine-based reversible modifications. The overall approach requires a simpler workflow with increased specificity compared to the commonly used biotinylation-based assays. The procedure for selective enrichment and analyses of S-nitrosylation 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 approximately 3 days for sample processing with an additional day for LC-MS/MS and data analysis.
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影响因子：
7
作者：
Murray, Christopher I.;Uhrigshardt, Helge;Van Eyk, Jennifer E.
通讯作者：
Van Eyk, Jennifer E.
影响因子：
3.9
作者：
Lind, C;Gerdes, R;Cotgreave, IA
通讯作者：
Cotgreave, IA
DOI：
10.1073/pnas.0707723105
发表时间：
2008-06-17
影响因子：
11.1
作者：
Leichert, Lars I.;Gehrke, Florian;Jakob, Ursula
通讯作者：
Jakob, Ursula
影响因子：
4.8
作者：
Forrester, Michael T.;Foster, Matthew W.;Stamler, Jonathan S.
通讯作者：
Stamler, Jonathan S.
影响因子：
6.6
作者：
Brandes N;Schmitt S;Jakob U
通讯作者：
Jakob U