Large-Scale
Capture of Peptides Containing Reversibly
Oxidized Cysteines by Thiol-Disulfide Exchange Applied to the Myocardial
Redox Proteome
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Abstract
Redox
regulation is emerging as an important post-translational
modification in cell signaling and pathogenesis. Cysteine (Cys) is
the most redox active of the commonly coded amino acids and is thus
an important target for redox-based modifications. Reactions that
oxidize the Cys sulfur atom to low oxidation states (e.g., disulfide)
are reversible, while further reactions to higher oxidation states
(e.g., sulfonic acid) may be irreversible under biological conditions.
Reversible modifications are particularly interesting as they mediate
redox signaling and regulation of proteins under physiological conditions
and during adaptation to oxidant stress. An enrichment method that
relied on rapid and specific alkylation of free Cys, followed by thiol-based
reduction and resin capture by thiol-disulfide exchange chemistry
was applied to isolate reversibly modified Cys-containing peptides.
Chromatographic conditions were optimized to provide increased specificity
by removal of noncovalent interactions. The technique was highly efficient,
based on near equimolar reactions with the resin, reproducible and
linear for peptide elution, as quantified by label-free mass spectrometry.
The method was applied to a complex protein lysate generated from
rat myocardial tissue and 6559 unique Cys-containing peptides from
2694 proteins were identified. Comparison with the rat database and
previous studies showed effective enrichment of proteins modified
by S-nitrosylation, disulfide formation, and Cys-sulfenic acid. Analysis
of amino acid sequence features indicated a preference for acidic
residues and increased hydrophilicity in the regions immediately up-
or downstream of the reactive Cys. This technique is ideally suited
for the enrichment and profiling of reversible Cys modifications on
a proteome-wide scale