Identifying Functional Cysteine Residues in the Mitochondria

The mitochondria are dynamic organelles that regulate oxidative metabolism and mediate cellular redox homeostasis. Proteins within the mitochondria are exposed to large fluxes in the surrounding redox environment. In particular, cysteine residues within mitochondrial proteins sense and respond to these redox changes through oxidative modifications of the cysteine thiol group. These oxidative modifications result in a loss in cysteine reactivity, which can be monitored using cysteine-reactive chemical probes and quantitative mass spectrometry (MS). Analysis of cell lysates treated with cysteine-reactive probes enable the identification of hundreds of cysteine residues, however, the mitochondrial proteome is poorly represented (S-nitrosation on mitochondrial proteins. Together, the mitochondrial cysteine enrichment strategy presented herein enables detailed characterization of protein modifications that occur within the mitochondria during (patho)­physiological fluxes in the redox environment.