Shotgun redox proteomics identifies specifically modified cysteines in key metabolic enzymes under oxidative stress in Saccharomyces cerevisiae
Shotgun redox proteomics identifies specifically modified cysteines in key metabolic enzymes under oxidative stress in Saccharomyces cerevisiae
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DOI：
10.1016/j.jprot.2009.01.023
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发表时间：
2009-05-02
影响因子：
3.3
通讯作者：
Antonio Barcena, Jose
中科院分区：
文献类型：
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作者：
McDonagh, Brian;Ogueta, Samuel;Antonio Barcena, Jose
硫醇基团的翻译后氧化还原修饰可以形成抗氧化保护和氧化还原控制的分子基础。我们已经实施了一种鸟枪式氧化还原蛋白质组技术来识别关键蛋白质中可逆氧化的精确半胱氨酸。将该方法应用于过氧化氢处理的酿酒酵母。通过共价氧化还原亲和层析的浓缩，可以分离出“氧化还原亚肽”，并用LC-MS/MS进行分析。含有特定可逆氧化半胱氨酸的独特多肽被用来鉴定对照和处理样品中的70多种蛋白质，其中27种蛋白质一致地存在于所有重复样品中。在大多数情况下，氧化还原修饰会对它们的功能产生负面影响，并减缓它们的代谢途径。数据的整合提供了与代谢防御策略一致的快照，通过氧化还原修饰调节关键酶，将能量重新引导到核酮糖-5-磷酸循环用于NADPH生产和抗氧化防御。这种普遍适用的方法使我们能够发现新的氧化还原调节蛋白质(DAHP和氨基甲酰磷酸合成酶，Doa1p)，并在许多已知的蛋白质中准确识别目标半胱氨酸。(C)2009爱思唯尔B.V.保留所有权利。
Postranslational redox modification of thiol groups can form the molecular basis of antioxidative protection and redox control. We have implemented a Shotgun redox proteomic technique to identify the precise cysteines reversibly oxidised in key proteins. The method was applied to Saccharomyces cerevisiae subjected to peroxide treatment. Enrichment by covalent redox affinity chromatography allowed the isolation of a "redox subpeptidome" that was analysed by LC-MS/MS. Unique peptides containing specific reversibly oxidised cysteines were used to identify over 70 proteins in control and treated samples of which 27 were consistently present in all replicates. In most cases, the redox modification negatively affects their function and slows down their metabolic pathways. integration of the data provides a snapshot consistent with a metabolic defensive strategy, regulating key enzymes by redox modification, redirecting energy toward ribulose-5-phosphate recycling for NADPH production and antioxidative defence.This generally applicable method has allowed us to discover new redox regulated proteins (DAHP and carbamoylphosphate synthases, Doa1p) and to precisely identify target cysteines in a number of known ones. (C) 2009 Elsevier B.V. All rights reserved.