title: Large-scale capture of peptides containing reversibly oxidized cysteines by thiol-disulfide exchange applied to the myocardial redox proteome.
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.
authors: School of Molecular Bioscience, The University of Sydney, Sydney, Australia.
authors: Jana
authors: J
authors: Paulech
authors: Nestor
authors: N
authors: Solis
authors: Alistair V G
authors: AV
authors: Edwards
authors: Max
authors: M
authors: Puckeridge
authors: Melanie Y
authors: MY
authors: White
authors: Stuart J
authors: SJ
authors: Cordwell
mesh_terms: D000818:Animals; D002845:Chromatography; D003545:Cysteine / Q000032:analysis*; D009206:Myocardium / Q000737:chemistry*; D010084:Oxidation-Reduction; D010455:Peptides / Q000032:analysis*; D020543:Proteome / Q000737:chemistry*; D040901:Proteomics; D051381:Rats; D011917:Rats, Inbred Lew; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D010455:Peptides; D020543:Proteome; D013438:Sulfhydryl Compounds; D003545:Cysteine