Bak Daniel W, Weerapana Eranthie
Department of Chemistry, Boston College, Chestnut Hill, MA, USA.
Methods Mol Biol. 2019;1967:211-227. doi: 10.1007/978-1-4939-9187-7_13.
Mitochondria are cellular sites of diverse redox biology, including ROS production, iron-sulfur biogenesis, and secondary metabolism, which all rely on proteogenic reactive cysteine residues. Mass spectrometry-based proteomic methods to monitor the reactivity and functionality of cysteine residues across complex proteomes have greatly expanded over the past decade. Here we describe a mitochondrial isolation procedure coupled with cysteine-reactive IA labeling that affords identification and characterization of functional mitochondrial cysteine residues that were heretofore inaccessible to whole-cell proteomic analysis.
线粒体是多种氧化还原生物学过程的细胞场所，包括活性氧生成、铁硫生物合成和次生代谢，这些过程都依赖于蛋白质ogenic反应性半胱氨酸残基。在过去十年中，基于质谱的蛋白质组学方法用于监测复杂蛋白质组中半胱氨酸残基的反应性和功能，得到了极大的发展。在这里，我们描述了一种线粒体分离程序，结合半胱氨酸反应性IA标记，可用于鉴定和表征功能性线粒体半胱氨酸残基，而这些残基在全细胞蛋白质组分析中以前是无法获得的。
