Protein S-nitrosylation is a cysteine post-translational modification mediated by nitric oxide. An increasing number of studies highlight S-nitrosylation as an important regulator of signaling involved in numerous cellular processes. Despite the significant progress in the development of redox proteomic methods, identification and quantification of endogeneous S-nitrosylation using high-throughput mass-spectrometry-based methods is a technical challenge because this modification is highly labile. To overcome this drawback, most methods induce S-nitrosylation chemically in proteins using nitrosylating compounds before analysis, with the risk of introducing nonphysiological S-nitrosylation. Here we present a novel method to efficiently identify endogenous S-nitrosopeptides in the macrophage total proteome. Our approach is based on the labeling of S-nitrosopeptides reduced by ascorbate with a cysteine specific phosphonate adaptable tag (CysPAT), followed by titanium dioxide (TiO₂) chromatography enrichment prior to nLC–MS/MS analysis. To test our procedure, we performed a large-scale analysis of this low-abundant modification in a murine macrophage cell line. We identified 569 endogeneous S-nitrosylated proteins compared with 795 following exogenous chemically induced S-nitrosylation. Importantly, we discovered 579 novel S-nitrosylation sites. The large number of identified endogenous S-nitrosylated peptides allowed the definition of two S-nitrosylation consensus sites, highlighting protein translation and redox processes as key S-nitrosylation targets in macrophages.

中文翻译：

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半胱氨酸特定的膦酸酯适应性标签结合TiO ₂色谱法表征巨噬细胞内源性S-亚硝基蛋白质组。

蛋白S-亚硝基化是由一氧化氮介导的半胱氨酸翻译后修饰。越来越多的研究表明，S-亚硝基化是众多细胞过程中重要的信号调节剂。尽管在氧化还原蛋白质组学方法的开发方面取得了重大进展，但是使用基于高通量质谱的方法鉴定和定量内源性S-亚硝基化仍是一项技术挑战，因为这种修饰非常不稳定。为了克服这一缺点，大多数方法在分析之前使用亚硝化化合物在蛋白质中诱导S-亚硝化，但存在引入非生理性S的风险。-亚硝基化。在这里，我们提出了一种新方法，可以有效地识别巨噬细胞总蛋白质组中的内源性S-亚硝基肽。我们的方法是基于抗坏血酸与半胱氨酸特异性膦酸酯适应性标签（CysPAT）还原的S-亚硝基肽的标记，然后在nLC-MS / MS分析之前进行二氧化钛（TiO ₂）色谱富集。为了测试我们的程序，我们对鼠巨噬细胞系中的这种低丰度修饰进行了大规模分析。我们鉴定了569种内源性S-亚硝基化蛋白，而外源化学诱导的S-亚硝化后为795种。重要的是，我们发现了579部小说S-亚硝基化位点。大量鉴定出的内源性S-亚硝基化肽可以定义两个S-亚硝基化的共有位点，突出了蛋白质翻译和氧化还原过程，这是巨噬细胞中关键的S-亚硝基化的靶标。