Identification and Quantification of S-Nitrosylation by Cysteine Reactive Tandem Mass Tag Switch Assay
Identification and Quantification of S-Nitrosylation by Cysteine Reactive Tandem Mass Tag Switch Assay
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DOI：
10.1074/mcp.m111.013441
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发表时间：
2012-02-01
影响因子：
7
通讯作者：
Van Eyk, Jennifer E.
中科院分区：
文献类型：
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作者：
Murray, Christopher I.;Uhrigshardt, Helge;Van Eyk, Jennifer E.
氧化还原开关是关键的半胱氨酸硫醇，其响应于细胞环境的变化而被修饰，从而赋予功能效应。S-亚硝基化（SNO）正在成为这些调节开关的重要调节剂;然而，关于这些特定半胱氨酸残基的性质以及如何解释氧化信号仍然未知。由于其不稳定的性质，通常使用生物素开关测定法检测SNO修饰。在此，使用一种新的同位素编码半胱氨酸巯基反应性多重试剂cysTMT（6）代替生物素，用于特异性检测SNO修饰和测定单个蛋白巯基反应性。采用cysTMT（6）定量转换分析结合质谱法测定了人肺动脉内皮细胞的S-亚硝基化。用S-亚硝基谷胱甘肽处理细胞裂解物，并用于鉴定179种蛋白质上的220个SNO修饰的半胱氨酸。使用这种方法，可以区分潜在的人为因素，包括蛋白质二硫键还原（6）和S-谷胱甘肽化（5）以及位点分配中模糊性的减少。在一系列NO-供体浓度（2，10，20 μ M; GSNO）的定量分析揭示了一个连续的反应，SNO-修饰。在活细胞中验证了半胱氨酸响应，证明随着氧化刺激的增加，更大数量的不太敏感的半胱氨酸残基被修饰。值得注意的是，发现大多数可用的半胱氨酸在当前处理中是未修饰的，这表明氧化修饰的显著额外能力。这些结果表明，细胞通过修饰的氧化还原开关的进行性和特异性积累来测量氧化刺激的大小的可能机制。Molecular & Cellular Proteomics 11：10.1074/mcp. M111.013441，1-12，2012.
Redox-switches are critical cysteine thiols that are modified in response to changes in the cell's environment conferring a functional effect. S-nitrosylation (SNO) is emerging as an important modulator of these regulatory switches; however, much remains unknown about the nature of these specific cysteine residues and how oxidative signals are interpreted. Because of their labile nature, SNO-modifications are routinely detected using the biotin switch assay. Here, a new isotope coded cysteine thiol-reactive multiplex reagent, cysTMT(6), is used in place of biotin, for the specific detection of SNO-modifications and determination of individual protein thiol-reactivity. S-nitrosylation was measured in human pulmonary arterial endothelia cells in vitro and in vivo using the cysTMT(6) quantitative switch assay coupled with mass spectrometry. Cell lysates were treated with S-nitrosoglutathione and used to identify 220 SNO-modified cysteines on 179 proteins. Using this approach it was possible to discriminate potential artifacts including instances of reduced protein disulfide bonds (6) and S-glutathionylation (5) as well as diminished ambiguity in site assignment. Quantitative analysis over a range of NO-donor concentrations (2, 10, 20 mu M; GSNO) revealed a continuum of reactivity to SNO-modification. Cysteine response was validated in living cells, demonstrating a greater number of less sensitive cysteine residues are modified with increasing oxidative stimuli. Of note, the majority of available cysteines were found to be unmodified in the current treatment suggesting significant additional capacity for oxidative modifications. These results indicate a possible mechanism for the cell to gauge the magnitude of oxidative stimuli through the progressive and specific accumulation of modified redox-switches. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.013441, 1-12, 2012.