S-Alkylating Labeling Strategy for Site-Specific Identification of the S-Nitrosoproteome
S-Alkylating Labeling Strategy for Site-Specific Identification of the S-Nitrosoproteome
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DOI：
10.1021/pr100680a
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发表时间：
2010-12-01
影响因子：
4.4
通讯作者：
Chen, Yu-Ju
中科院分区：
文献类型：
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作者：
Chen, Yi-Ju;Ku, Wei-Chi;Chen, Yu-Ju
S亚硝化是一种由一氧化氮引起的半胱氨酸残基的翻译后修饰，它介导了许多生理功能。由于S亚硝化的不稳定性质，质谱仪(MS)的检测是具有挑战性的。本文利用S亚硝半胱氨酸上的不可逆生物素化建立了S烷基化标记策略，利用不含内源性一氧化氮合酶的COS7细胞进行LC-MS/MS定位鉴定S亚硝基蛋白质组。我们证明了S烷基化标记策略显著提高了游离半胱氨酸的阻断效率，最大限度地减少了二硫键交换引起的假阳性鉴定，并提高了消化效率。我们在S-亚硝基-N-乙酰青霉胺/L-半胱氨酸处理的小鼠MS-1血管内皮细胞中鉴定了与384种蛋白质相对应的586个唯一的S亚硝化位点。包括2 34个未见报道的S亚硝化蛋白质。进一步分析已鉴定的84个跨膜蛋白的拓扑结构，发现它们的S亚硝化位点大多面向细胞质一侧，这意味着除了已知的酸/碱基序外，S亚硝化还发生在细胞质中。推导出的10个共识基序表明，局部疏水性和三级结构中的酸/碱基序的结合有助于S亚硝化的专一性。此外，S亚硝化的半胱氨酸显示出对β链的偏好，S亚硝半胱氨酸的相对表面可及性较低
S-nitrosylation, a post-translational modification of cysteine residues induced by nitric oxide, mediates many physiological functions Due to the labile nature of S nitrosylation, detection by mass spectrometry (MS) is challenging Here we developed an S-alkylating labeling strategy using the irreversible biotinylation on S-nitrosocysteines for site-specific identification of the S-nitrosoproteome by LC-MS/MS Using COS-7 cells without endogenous nitric oxide synthase, we demonstrated that the S-alkylating labeling strategy substantially improved the blocking efficiency of free cysteines minimized the false-positive identification caused by disulfide interchange and increased the digestion efficiency for improved peptide identification using MS analyses Using this strategy, we identified total 586 unique S-nitrosylation sites corresponding to 384 proteins in S-nitroso-N-acetylpenicillamine (SNAP)/L-cysteine-treated mouse MS-1 endothelial cells, including 234 previously unreported S-nitrosylated proteins When the topologies of 84 identified transmembrane proteins were further analyzed their S-nitrosylation sites were found to mostly face the cytoplasmic side implying that S-nitrosylation occurs in the cytoplasm In addition to the previously known acid/basic motifs the ten deduced consensus motifs suggested that combination of local hydrophobicity and acid/base motifs in the tertiary structure contribute to the specificity of S-nitrosylation Moreover the S-nitrosylated cysteines showed preference on beta-strand having lower relative surface accessibility at the S-nitrosocysteines