title: Global analysis of myocardial peptides containing cysteines with irreversible sulfinic and sulfonic acid post-translational modifications.
abstract: Cysteine (Cys) oxidation is a crucial post-translational modification (PTM) associated with redox signaling and oxidative stress. As Cys is highly reactive to oxidants it forms a range of post-translational modifications, some that are biologically reversible (e.g. disulfides, Cys sulfenic acid) and others (Cys sulfinic [Cys-SO2H] and sulfonic [Cys-SO3H] acids) that are considered "irreversible." We developed an enrichment method to isolate Cys-SO2H/SO3H-containing peptides from complex tissue lysates that is compatible with tandem mass spectrometry (MS/MS). The acidity of these post-translational modification (pKa Cys-SO3H < 0) creates a unique charge distribution when localized on tryptic peptides at acidic pH that can be utilized for their purification. The method is based on electrostatic repulsion of Cys-SO2H/SO3H-containing peptides from cationic resins (i.e. "negative" selection) followed by "positive" selection using hydrophilic interaction liquid chromatography. Modification of strong cation exchange protocols decreased the complexity of initial flowthrough fractions by allowing for hydrophobic retention of neutral peptides. Coupling of strong cation exchange and hydrophilic interaction liquid chromatography allowed for increased enrichment of Cys-SO2H/SO3H (up to 80%) from other modified peptides. We identified 181 Cys-SO2H/SO3H sites from rat myocardial tissue subjected to physiologically relevant concentrations of H2O2 (<100 μm) or to ischemia/reperfusion (I/R) injury via Langendorff perfusion. I/R significantly increased Cys-SO2H/SO3H-modified peptides from proteins involved in energy utilization and contractility, as well as those involved in oxidative damage and repair.
authors.affiliation: From the ‡School of Molecular Bioscience, The University of Sydney, Australia 2006;
authors.forename: Jana
authors.initials: J
authors.lastname: Paulech
authors.affiliation: From the ‡School of Molecular Bioscience, The University of Sydney, Australia 2006; §Charles Perkins Centre, The University of Sydney, Australia 2006;
authors.forename: Kiersten A
authors.initials: KA
authors.lastname: Liddy
authors.affiliation: ¶Children's Medical Research Institute, Westmead, Australia 2145; ‖Centre for Clinical Proteomics, Odense University Hospital, Odense C, Denmark DK-5000; **Department of Biochemistry and Molecular Biology, University of Southern Denmark, Denmark DK-5230;
authors.forename: Kasper
authors.initials: K
authors.lastname: Engholm-Keller
authors.affiliation: From the ‡School of Molecular Bioscience, The University of Sydney, Australia 2006; §Charles Perkins Centre, The University of Sydney, Australia 2006; ‡‡Discipline of Pathology, School of Medical Sciences, The University of Sydney, Australia 2006.
authors.forename: Melanie Y
authors.initials: MY
authors.lastname: White
authors.affiliation: From the ‡School of Molecular Bioscience, The University of Sydney, Australia 2006; §Charles Perkins Centre, The University of Sydney, Australia 2006; ‡‡Discipline of Pathology, School of Medical Sciences, The University of Sydney, Australia 2006 stuart.cordwell@sydney.edu.au.
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Cordwell
mesh_terms: D000818:Animals; D003545:Cysteine / Q000302:isolation & purification*; D009206:Myocardium / Q000378:metabolism* / Q000473:pathology; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification; D051381:Rats; D011917:Rats, Inbred Lew; D015427:Reperfusion Injury / Q000209:etiology / Q000473:pathology; D055672:Static Electricity; D013441:Sulfinic Acids / Q000737:chemistry; D013451:Sulfonic Acids / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D010455:Peptides; D020543:Proteome; D013441:Sulfinic Acids; D013451:Sulfonic Acids; D003545:Cysteine