Cysteine, the most nucleophilic amino acid, has a number of important functions within the protein, from catalysis to redox signaling and disulfide bond formation. Thus, mutation and misregulation of these crucial residues can lead to variety of disease states, including cancer, aging, and other degenerative diseases. Oxidative modification of cysteine by endogenous reactive oxygen species (ROS) can result in downstream signaling events and shifts in gene expression and cellular metabolism. As mitochondria are a major source of cellular ROS, it is important to have platforms that can specifically monitor cysteine oxidation of mitochondrial localized proteins. In previous whole cell proteomics studies, cysteine reactivity and sensitivity to oxidative stress have been assayed through the use of cysteine reactive probes, such as iodoacetamide (IA)-alkyne, and quantitative mass spectrometry (MS). Yet these studies have generally displayed poor coverage of the mitochondrial proteome. Here, we have developed and implemented a novel proteomic platform to improve MS identification of low abundance mitochondrial cysteine residues, through mitochondrial isolation and subsequent IA-alkyne labeling, enrichment, and MS identification of mitochondrial reactive cysteine containing peptides. Using this platform, more than 1,500 reactive cysteine containing peptides from ~450 mitochondrial proteins could be interrogated, compared to the ~100 mitochondrial proteins identified in whole cell experiments. This platform allowed for the ranking of mitochondrial cysteine residues by both reactivity and sensitivity to undergo S-nitrosation, an oxidative cysteine post-translational modification. Currently, this platform is being used to investigate mitochondrial cysteine redox signaling in breast cancer, in order to understand of the role of relationship between ROS signaling and tumor progression.<br/>

Chemoproteomic methods for investigating mitochondrial cysteine oxidation in disease

255th National Meeting (2018)

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