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
Department of Pathology and Anatomical Sciences, ‡Department of Biochemistry, and §Center for Translational Neuroscience, University of Missouri School of Medicine , Columbia, Missouri, United States.
J Proteome Res. 2014 Jul 3;13(7):3200-11. doi: 10.1021/pr401179v. Epub 2014 Jun 13.
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.
S-亚硝基化是一种基于氧化还原的蛋白质翻译后修饰，响应一氧化氮信号，参与广泛的生物过程。由于该修饰的不稳定性和低丰度，蛋白质S-亚硝基化的检测和定量一直是具有挑战性的任务。许多研究使用基于质谱（MS）的方法，结合不同的硫醇反应试剂来标记和鉴定具有S-亚硝基化半胱氨酸（SNO-Cys）的蛋白质。在本研究中，我们开发了一种新型的碘代TMT转换分析法（ISA），使用一组等压的硫醇反应性碘代TMT六重试剂来特异性检测和定量蛋白质S-亚硝基化。用碘代TMT六重试剂对SNO-Cys进行不可逆标记，能够对S-亚硝基化蛋白质进行免疫亲和检测，通过抗TMT树脂富集碘代TMT标记的肽段，重要的是，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，我们通过竞争性洗脱显著提高了抗TMT肽段的富集效率。使用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，并揭示了大蒜中的S-烯丙基半胱氨酸对LPS诱导的抗氧化信号和线粒体代谢途径中蛋白质S-亚硝基化的影响。ISA被证明是一种用于复杂样品中S-亚硝基化定量分析的有效蛋白质组学方法，将有助于阐明疾病中氧化应激的分子机制。
