Identification of S-glutathionylated cellular proteins during oxidative stress and constitutive metabolism by affinity purification and proteomic analysis
Identification of S-glutathionylated cellular proteins during oxidative stress and constitutive metabolism by affinity purification and proteomic analysis
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DOI：
10.1016/s0003-9861(02)00468-x
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发表时间：
2002-10-15
影响因子：
3.9
通讯作者：
Cotgreave, IA
中科院分区：
文献类型：
--
作者：
Lind, C;Gerdes, R;Cotgreave, IA
蛋白质的氧化还原修饰被认为在调节细胞功能中发挥核心作用。然而，缺乏用于分析复杂混合物中单个蛋白质氧化还原状态的高通量技术。因此，我们的目标是开发一种合适的技术来快速识别通过 S-谷胱甘肽化进行关键硫醇氧化的蛋白质。该方法基于谷氧还蛋白对混合二硫化物的特异性还原、它们与N-乙基马来酰亚胺-生物素的反应、标记蛋白的亲和纯化以及通过蛋白质组分析进行鉴定。该方法明确鉴定了 43 种用于 S-谷胱甘肽化的新型细胞蛋白底物。这些包括蛋白质伴侣、细胞骨架蛋白、细胞周期调节剂和中间代谢酶。对从经历二酰胺诱导的氧化应激和组成代谢期间的细胞中提取的 S-谷胱甘肽化蛋白质的模式进行比较，揭示了常见蛋白质底物和在氧化应激期间未能经历增强的 S-谷胱甘肽化的底物。化学标记、选择和识别 S-谷胱甘肽化蛋白质的能力，特别是在组成性代谢过程中，将大大增强建立细胞蛋白质翻译后氧化还原修饰作为协调细胞功能的重要生化控制机制的努力。 (C) 2002 年爱思唯尔科学（美国）。版权所有。
Redox modification of proteins is proposed to play a central role in regulating cellular function. However, high-throughput techniques for the analysis of the redox status of individual proteins in complex mixtures are lacking. The aim was thus to develop a suitable technique to rapidly identify proteins undergoing oxidation of critical thiols by S-glutathionylation. The method is based on the specific reduction of mixed disulfides by glutaredoxin, their reaction with N-ethylmaleimitle-biotin, affinity purification of tagged proteins, and identification by proteomic analysis. The method unequivocally identified 43 mostly novel cellular protein substrates for S-glutathionylation. These include protein chaperones, cytoskeletal proteins, cell cycle regulators, and enzymes of intermediate metabolism. Comparisons of the patterns of S-glutathionylated proteins extracted from cells undergoing diamide-induced oxidative stress and during constitutive metabolism reveal both common protein substrates and substrates failing to undergo enhanced S-glutathionylation during oxidative stress. The ability to chemically tag, select, and identify S-glutathionylated proteins, particularly during constitutive metabolism, will greatly enhance efforts to establish posttranslational redox modification of cellular proteins as an important biochemical control mechanism in coordinating cellular function. (C) 2002 Elsevier Science (USA). All rights reserved.