Pan Kuan-Ting, Chen Yi-Yun, Pu Tsung-Hsien, Chao Yu-Shu, Yang Chun-Yi, Bomgarden Ryan D, Rogers John C, Meng Tzu-Ching, Khoo Kay-Hooi
1 Institute of Biochemical Sciences, National Taiwan University , Taipei, Taiwan .
Antioxid Redox Signal. 2014 Mar 20;20(9):1365-81. doi: 10.1089/ars.2013.5326. Epub 2013 Oct 23.
Distinctive states of redox-dependent cysteine (Cys) modifications are known to regulate signaling homeostasis under various pathophysiological conditions, including myocardial injury or protection in response to ischemic stress. Recent evidence further implicates a dynamic interplay among these modified forms following changes in cellular redox environment. However, a precise delineation of multiplexed Cys modifications in a cellular context remains technically challenging. To this end, we have now developed a mass spectrometry (MS)-based quantitative approach using a set of novel iodoacetyl-based Cys-reactive isobaric tags (irreversible isobaric iodoacetyl Cys-reactive tandem mass tag [iodoTMT]) endowed with unique irreversible Cys-reactivities.
We have established a sequential iodoTMT-switch procedure coupled with efficient immunoenrichment and advanced shotgun liquid chromatography-MS/MS analysis. This workflow allows us to differentially quantify the multiple redox-modified forms of a Cys site in the original cellular context. In one single analysis, we have identified over 260 Cys sites showing quantitative differences in multiplexed redox modifications from the total lysates of H9c2 cardiomyocytes experiencing hypoxia in the absence and presence of S-nitrosoglutathione (GSNO), indicative of a distinct pattern of individual susceptibility to S-nitrosylation or S-glutathionylation. Among those most significantly affected are proteins functionally implicated in hypoxic damage from which we showed that GSNO would protect.
We demonstrate for the first time how quantitative analysis of various Cys-redox modifications occurring in biological samples can be performed precisely and simultaneously at proteomic levels.
We have not only developed a new approach to map global Cys-redoxomic regulation in vivo, but also provided new evidences implicating Cys-redox modifications of key molecules in NO-mediated ischemic cardioprotection.
已知氧化还原依赖性半胱氨酸（Cys）修饰的不同状态可在各种病理生理条件下调节信号稳态，包括心肌损伤或对缺血应激的保护反应。最近的证据进一步表明，在细胞氧化还原环境变化后，这些修饰形式之间存在动态相互作用。然而，在细胞环境中精确描绘多重Cys修饰在技术上仍然具有挑战性。为此，我们现在开发了一种基于质谱（MS）的定量方法，该方法使用一组新型的基于碘乙酰基的Cys反应性等压标签（不可逆等压碘乙酰基Cys反应性串联质量标签 [iodoTMT]），具有独特的不可逆Cys反应性。
我们建立了一种顺序碘代TMT转换程序，结合高效免疫富集和先进的鸟枪法液相色谱 - 质谱/质谱分析。该工作流程使我们能够在原始细胞环境中差异定量Cys位点的多种氧化还原修饰形式。在一次分析中，我们从在不存在和存在S-亚硝基谷胱甘肽（GSNO）的情况下经历缺氧的H9c2心肌细胞的总裂解物中鉴定出超过260个Cys位点，这些位点在多重氧化还原修饰中显示出定量差异，表明对S-亚硝基化或S-谷胱甘肽化的个体敏感性存在明显模式。其中受影响最显著的是与缺氧损伤功能相关的蛋白质，我们证明GSNO可以对其起到保护作用。
我们首次展示了如何在蛋白质组学水平上精确且同时地对生物样品中发生的各种Cys-氧化还原修饰进行定量分析。
我们不仅开发了一种在体内绘制全局Cys-氧化还原组调控的新方法，还提供了新的证据，表明关键分子的Cys-氧化还原修饰在NO介导的缺血性心脏保护中起作用。
