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. 2015 Mar;14(3):609-20.
doi: 10.1074/mcp.M114.044347. Epub 2015 Jan 5.

Global analysis of myocardial peptides containing cysteines with irreversible sulfinic and sulfonic acid post-translational modifications

Jana Paulech  1 , Kiersten A Liddy  2 , Kasper Engholm-Keller  3 , Melanie Y White  4 , Stuart J Cordwell  5
Affiliations

Global analysis of myocardial peptides containing cysteines with irreversible sulfinic and sulfonic acid post-translational modifications

Jana Paulech et al. Mol Cell Proteomics. 2015 Mar.

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.

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Figures

Fig. 1.
Fig. 1.
Resolution of Cys-SO3H peptides from performic oxidized myocardial extract by SCX. A, In-solution charge distributions of peptides across the six summed SCX fractions, plotted as percent of PSMs for each fraction; B, Unique peptides as a percent of total identifications occurring in all fractions, plotted for total peptides and Cys-SO3H peptides, across the six SCX fractions; C, Cys-SO3H, pyroglutamate, acetyl-modified, and unmodified peptides as a percent of total identified peptides in each fraction.
Fig. 2.
Fig. 2.
HILIC separation positively selects for Cys-SO3H-containing peptides post-SCX negative selection. A, Percent total PSMs arising from unmodified/modified peptides with increasing aqueous phase; B, percentage of total Cys-SO3H peptide identifications arising from FT, MeCN, and 10% KCl fractions following HILIC enrichment. Inset shows the subdivision of peptides contained within the 10% KCl fraction by whether they are His-containing or contain a missed cleavage/(R/K)-P site.
Fig. 3.
Fig. 3.
Schematic of SCX-HILIC method for the enrichment of irreversibly oxidized Cys (oxCys)-containing peptides. Initial binding of peptide samples by SCX chromatography in an aqueous buffer allows for electrostatic repulsion of peptides containing oxCys and their negative selection into the FT fraction. Subsequent washing with an aqueous phase containing an organic modifier will liberate hydrophobic and neutral oxCys peptides retained by the resin along with an increasing amount of neutral non-oxCys peptides (e.g. PTM peptides). Elution with a low salt concentration will elute retained oxCys peptides containing basic residues (H/K/R), as well as singly charged tryptic peptides, whereas increased salt concentrations will elute increasingly positive tryptic peptides. oxCys peptides from the initial fractions may be positively selected by HILIC chromatography where they are retained later than more hydrophobic PTM peptides.
Fig. 4.
Fig. 4.
Fractionation of peptides generated from myocardial extract oxidized by 10 mm H2O2. A, Percent total unique and unique Cys-SO2H/SO3H peptides identified in each fraction; B, PSMs corresponding to Cys-SO2H/SO3H peptides enriched with increased aqueous phase by HILIC, with the percent total representing the average over all fractions.
Fig. 5.
Fig. 5.
Cys-SO2H/SO3H-containing peptide identifications in myocardial extracts oxidized by differing [H2O2] and in myocardial tissue subjected to Langendorff perfusion (NITC and I/R). A, Number of sites observed in each sample, with total sites, Cys-SO2H and Cys-SO3H sites denoted; B, overlap of Cys-SO2H/SO3H sites identified at 100 nm, 10 μm, and 100 μm H2O2.
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