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-SO 2 H] and sulfonic [Cys-SO 3 H] acids) that are considered "irreversible." We developed an enrichment method to isolate Cys-SO 2 H/SO 3 H-containing peptides from complex tissue lysates that is compatible with tandem mass spectrometry (MS/MS). The acidity of these post-translational modification (pK a Cys-SO 3 H < 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-SO 2 H/SO 3 H-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-SO 2 H/SO 3 H (up to 80%) from other modified peptides. We identified 181 Cys-SO 2 H/SO 3 H sites from rat myocardial tissue subjected to physiologically relevant concentrations of H 2 O 2 (<100 M) or to ischemia/reperfusion (I/R) injury via Langendorff perfusion.

High-throughput screening of cellular redox sensors using modern redox proteomics approaches

“…Using this approach, reduced and oxidized form of cysteines can be simultaneously identified as different alkylating reagents can be differentiated by MS [94]. In addition to identify reversible oxidative modifications, a scheme to isolate sulfinylated (Pr-SO 2 H) and sulfonylated (Pr-SO 3 H) thiols has been developed [95]. Using this method, 181 Cys-SO 2 H/ÀSO 3 H peptides were identified after ischemia/ reperfusion injury in rat myocardial tissue [95].…”

“…In addition to identify reversible oxidative modifications, a scheme to isolate sulfinylated (Pr-SO 2 H) and sulfonylated (Pr-SO 3 H) thiols has been developed [95]. Using this method, 181 Cys-SO 2 H/ÀSO 3 H peptides were identified after ischemia/ reperfusion injury in rat myocardial tissue [95].…”

Quantitative proteomic characterization of redox-dependent post-translational modifications on protein cysteines

“…Multi-dimensional fractionation and high-resolution separation strategies could be useful in reducing sample complexity and enhancing the proteome coverage and sensitivity of detection. 17, 113, 124 …”

“…However, irreversible oxidation, such as SO 2 H and SO 3 H, are typically excluded in such measurements since the contribution of irreversible oxidation is often minor in most conditions. 17 In addition, occupancy analysis is not amenable to peptides containing multiple Cys residues due to the existence of multiple combinations of oxidized cysteines in a given sequence. Despite these limitations, a number of reported approaches have proved to be quite effective in measuring site occupancy for different forms of PTMs and for identifying functional Cys sites.…”

“…Thus, direct analysis of these modifications in a complex sample is conceptually feasible. Chromatography-based methods, such as ion exchange chromatography (IEX) 16, 17 and hydrophilic interaction chromatography (HILIC) 17 , have been used to enhance the proteomic discovery of these modifications. In contrast, measurement of reversible PTMs in complex biological systems has been challenging for several reasons, including the labile nature of these modifications, artificial oxidation, and conversion between different forms of reversible PTMs during sample processing.…”