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 (<10% of identified
peptides), due to the low abundance of mitochondrial proteins and
suppression of mitochondrial peptide MS signals by highly abundant
cytosolic peptides. Here, we apply a mitochondrial isolation and purification
protocol to substantially increase coverage of the mitochondrial cysteine
proteome. Over 1500 cysteine residues from ∼450 mitochondrial
proteins were identified, thereby enabling interrogation of an unprecedented
number of mitochondrial cysteines. Specifically, these mitochondrial
cysteines were ranked by reactivity to identify hyper-reactive cysteines
with potential catalytic and regulatory functional roles. Furthermore,
analyses of mitochondria exposed to nitrosative stress revealed previously
uncharacterized sites of protein 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.