Use of dimedone-based chemical probes for sulfenic acid detection methods to visualize and identify labeled proteins.
Use of dimedone-based chemical probes for sulfenic acid detection methods to visualize and identify labeled proteins.
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使用二二酮的化学探针用于硫酸检测方法，以可视化和鉴定标记的蛋白质。
DOI：
10.1016/s0076-6879(10)73004-4
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发表时间：
2010
影响因子：
--
通讯作者：
Poole, Leslie B.
中科院分区：
文献类型：
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作者：
Nelson, Kimberly J.;Klomsiri, Chananat;Codreanu, Simona G.;Soito, Laura;Liebler, Daniel C.;Rogers, LeAnn C.;Daniel, Larry W.;Poole, Leslie B.
可逆的硫醇修饰是活性氧调节细胞信号通路的主要组成部分。过氧化氢、过氧亚硝酸盐或脂质过氧化氢都能氧化半胱氨酸形成半胱氨酸磺酸；这种反应中间体可以被硫氧还蛋白等细胞还原剂直接还原为硫醇，或者进一步参与与同一或另一种蛋白质中的谷胱甘肽或半胱氨酸残基形成二硫键。为了确定半胱氨酸修饰的直接蛋白质靶标及其氧化条件，已开发了一系列连接到亲和标记或荧光标记的双酮试剂，这些试剂专门烷基化和捕获半胱氨酸磺酸。在本章中，我们提供了使用我们的生物素标记试剂之一DCP-Bio1来识别和监测在体外和体内被氧化的蛋白质的详细方法。利用链霉亲和素连接的琼脂糖珠，这种生物素连接的试剂可以用来亲和捕获标记的蛋白质。在洗脱前对珠子进行严格的洗涤，可通过共免疫沉淀或非特异性结合将未标记蛋白质对浓缩物质的污染降至最低。特别是，我们建议在其中一个洗涤剂中加入DTT，以去除通过二硫键与生物素化蛋白质共价连接的蛋白质，除非这些连接的蛋白质是感兴趣的。我们还提供了监测单个蛋白质中半胱氨酸氧化的有针对性的方法，跟踪细胞中总半胱氨酸氧化的全球方法，以及识别具有氧化还原敏感半胱氨酸的新蛋白质的蛋白质组学分析指南。
Reversible thiol modification is a major component of the modulation of cell-signaling pathways by reactive oxygen species. Hydrogen peroxide, peroxynitrite, or lipid hydroperoxides are all able to oxidize cysteines to form cysteine sulfenic acids; this reactive intermediate can be directly reduced to thiol by cellular reductants such as thioredoxin or further participate in disulfide bond formation with glutathione or cysteine residues in the same or another protein. To identify the direct protein targets of cysteine modification and the conditions under which they are oxidized, a series of dimedone-based reagents linked to affinity or fluorescent tags have been developed that specifically alkylate and trap cysteine sulfenic acids. In this chapter, we provide detailed methods using one of our biotin-tagged reagents, DCP-Bio1, to identify and monitor proteins that are oxidized in vitro and in vivo. Using streptavidin-linked agarose beads, this biotin-linked reagent can be used to affinity capture labeled proteins. Stringent washing of the beads prior to elution minimizes the contamination of the enriched material with unlabeled proteins through coimmunoprecipitation or nonspecific binding. In particular, we suggest including DTT in one of the washes to remove proteins covalently linked to biotinylated proteins through a disulfide bond, except in cases where these linked proteins are of interest. We also provide methods for targeted approaches monitoring cysteine oxidation in individual proteins, global approaches to follow total cysteine oxidation in the cell, and guidelines for proteomic analyses to identify novel proteins with redox sensitive cysteines.