Department of Pediatrics, Children's Hospital of Philadelphia Research Institute, Philadelphia, PA 19104, USA.
Methods. 2013 Aug 1;62(2):165-70. doi: 10.1016/j.ymeth.2012.10.009. Epub 2012 Oct 29.
Protein S-nitrosylation is considered as one of the molecular mechanisms by which nitric oxide regulates signaling events and protein function. The present review presents an updated method which allows for the site-specific detection of S-nitrosylated proteins in vivo. The method is based on enrichment of S-nitrosylated proteins or peptides using organomercury compounds followed by LC-MS/MS detection. Technical aspects for determining the reaction and binding efficiency of the mercury resin that assists enrichment of S-nitrosylated proteins are presented and discussed. In addition, emphasis is given to the specificity of the method by providing technical details for the generation of four chemically distinct negative controls. Finally it is provided an overview of the key steps for generation and evaluation of mass spectrometry derived data.
蛋白质 S-亚硝基化被认为是一氧化氮调节信号事件和蛋白质功能的分子机制之一。本综述介绍了一种新的方法，该方法可在体内特异性检测 S-亚硝基化蛋白质。该方法基于使用有机汞化合物富集 S-亚硝基化蛋白质或肽，然后进行 LC-MS/MS 检测。本文介绍并讨论了确定协助 S-亚硝基化蛋白质富集的汞树脂反应和结合效率的技术方面。此外，通过提供四个化学性质不同的阴性对照的生成技术细节，强调了该方法的特异性。最后，本文概述了生成和评估质谱衍生数据的关键步骤。
