Proteome-wide detection of S-nitrosylation targets and motifs using bioorthogonal cleavable-linker-based enrichment and switch technique
Proteome-wide detection of S-nitrosylation targets and motifs using bioorthogonal cleavable-linker-based enrichment and switch technique
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DOI：
10.1038/s41467-019-10182-4
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发表时间：
2019-05-16
影响因子：
16.6
通讯作者：
Zahedi, Rene P.
中科院分区：
文献类型：
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作者：
Mnatsakanyan, Ruzanna;Markoutsa, Stavroula;Zahedi, Rene P.
半胱氨酸修饰成为细胞信号传导和体内平衡的重要参与者。在这里，我们提出了一个化学蛋白质组学的策略，定量分析可逆修饰的半胱氨酸使用生物正交可切割连接和开关技术（Cys-BOOST）。与iodoTMT相比，Cys-BOOST的灵敏度高出三倍，特异性和精确度也相当高。分析S-亚硝基谷胱甘肽（GSNO）处理和未处理的HeLa提取物中的S-亚硝基化（SNO）Cys-BOOST在3，632种蛋白质上鉴定了8，304个SNO位点，覆盖了蛋白质组的广泛动态范围。具有不同GSNO反应性的SNO位点的一致基序证实了用于NO靶向特定半胱氨酸的酸碱催化和局部疏水性两者的相关性。将Cys-BOOST应用于SH-SY 5 Y细胞，我们在基础条件下鉴定了2，151个SNO位点，并揭示了作为对早期亚硝化应激的响应的显著变化的SNO水平，涉及神经元（轴突）发生，突触传递，蛋白质折叠/翻译和DNA复制。我们的工作表明，SNO作为一个全球性的蛋白质功能调节类似于磷酸化和泛素化。
Cysteine modifications emerge as important players in cellular signaling and homeostasis. Here, we present a chemical proteomics strategy for quantitative analysis of reversibly modified Cysteines using bioorthogonal cleavable-linker and switch technique (Cys-BOOST). Compared to iodoTMT for total Cysteine analysis, Cys-BOOST shows a threefold higher sensitivity and considerably higher specificity and precision. Analyzing S-nitrosylation (SNO) in S-nitrosoglutathione (GSNO)-treated and non-treated HeLa extracts Cys-BOOST identifies 8,304 SNO sites on 3,632 proteins covering a wide dynamic range of the proteome. Consensus motifs of SNO sites with differential GSNO reactivity confirm the relevance of both acid-base catalysis and local hydrophobicity for NO targeting to particular Cysteines. Applying Cys-BOOST to SH-SY5Y cells, we identify 2,151 SNO sites under basal conditions and reveal significantly changed SNO levels as response to early nitrosative stress, involving neuro(axono) genesis, glutamatergic synaptic transmission, protein folding/translation, and DNA replication. Our work suggests SNO as a global regulator of protein function akin to phosphorylation and ubiquitination.