Proteomic quantification and site-mapping of S-nitrosylated proteins using isobaric iodoTMT reagents.
Proteomic quantification and site-mapping of S-nitrosylated proteins using isobaric iodoTMT reagents.
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DOI：
10.1021/pr401179v
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发表时间：
2014-07-03
影响因子：
4.4
通讯作者：
Gu, Zezong
中科院分区：
文献类型：
--
作者：
Qu, Zhe;Meng, Fanjun;Bomgarden, Ryan D.;Viner, Rosa I.;Li, Jilong;Rogers, John C.;Cheng, Jianlin;Greenlief, C. Michael;Cui, Jiankun;Lubahn, Dennis B.;Sun, Grace Y.;Gu, Zezong
s -亚硝基化是一种基于氧化还原的蛋白质翻译后修饰，是对一氧化氮信号的反应，涉及广泛的生物过程。由于蛋白质s -亚硝基化修饰的不稳定性和低丰度，检测和定量一直是一项具有挑战性的任务。许多研究使用基于质谱（MS）的方法和不同的硫醇反应试剂来标记和鉴定含有s -亚硝基化半胱氨酸（SNO-Cys）的蛋白质。在这项研究中，我们开发了一种新的碘dotmt开关试验（ISA），使用一组等压的硫醇反应性碘tmtsixplex试剂来特异性检测和定量蛋白质s -亚硝基化。用iodoTMTsixplex试剂对SNO-Cys进行不可逆标记，可以对s -亚硝基化蛋白进行免疫亲和检测，通过抗tmt树脂富集iodotmt标记的肽，更重要的是，可以通过液相色谱-串联质谱进行明确的修饰位点定位和多重定量。此外，通过竞争洗脱，我们显著提高了抗tmt肽的富集效率。利用ISA，我们在小鼠BV-2小胶质细胞中鉴定了一组响应脂多糖（LPS）刺激的SNO-Cys位点，并揭示了大蒜s -烯丙基半胱氨酸对LPS诱导的抗氧化信号通路和线粒体代谢途径中蛋白质s -亚硝基化的影响。ISA被证明是一种有效的蛋白质组学方法，可以定量分析复杂样品中的s -亚硝基化，并有助于阐明疾病中亚硝基化胁迫的分子机制。
S-Nitrosylation is a redox-based protein post-translational modification in response to nitric oxide signaling and is involved in a wide range of biological processes. Detection and quantification of protein S-nitrosylation have been challenging tasks due to instability and low abundance of the modification. Many studies have used mass spectrometry (MS)-based methods with different thiol-reactive reagents to label and identify proteins with S-nitrosylated cysteine (SNO-Cys). In this study, we developed a novel iodoTMT switch assay (ISA) using an isobaric set of thiol-reactive iodoTMTsixplex reagents to specifically detect and quantify protein S-nitrosylation. Irreversible labeling of SNO-Cys with the iodoTMTsixplex reagents enables immune-affinity detection of S-nitrosylated proteins, enrichment of iodoTMT-labeled peptides by anti-TMT resin, and importantly, unambiguous modification site-mapping and multiplex quantification by liquid chromatography–tandem MS. Additionally, we significantly improved anti-TMT peptide enrichment efficiency by competitive elution. Using ISA, we identified a set of SNO-Cys sites responding to lipopolysaccharide (LPS) stimulation in murine BV-2 microglial cells and revealed effects of S-allyl cysteine from garlic on LPS-induced protein S-nitrosylation in antioxidative signaling and mitochondrial metabolic pathways. ISA proved to be an effective proteomic approach for quantitative analysis of S-nitrosylation in complex samples and will facilitate the elucidation of molecular mechanisms of nitrosative stress in disease.
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影响因子：
4.4
作者：
Chen, Yi-Ju;Ku, Wei-Chi;Chen, Yu-Ju
通讯作者：
Chen, Yu-Ju
影响因子：
20.3
作者：
Everts, Bart;Amiel, Eyal;Pearce, Edward J.
通讯作者：
Pearce, Edward J.
DOI：
10.1073/pnas.1008036107
发表时间：
2010-09-28
影响因子：
11.1
作者：
Doulias, Paschalis-Thomas;Greene, Jennifer L.;Ischiropoulos, Harry
通讯作者：
Ischiropoulos, Harry
影响因子：
5.1
作者：
Gu Z;Nakamura T;Lipton SA
通讯作者：
Lipton SA
影响因子：
7.3
作者：
Doulias PT;Tenopoulou M;Greene JL;Raju K;Ischiropoulos H
通讯作者：
Ischiropoulos H