Novel Oxidative Modifications in Redox-Active Cysteine Residues

Abstract

Redox-active cysteine, a highly reactive sulfhydryl, is one of the major targets of ROS. Formation of disulfide bonds and other oxidative derivatives of cysteine including sulfenic, sulfinic, and sulfonic acids, regulates the biological function of various proteins. We identified novel lowabundant cysteine modifications in cellular GAPDH purified on 2-dimensional gel electrophoresis (2D-PAGE) by employing selectively excluded mass screening analysis for nano ultraperformance liquid chromatography-electrospray-quadrupole-time of flight tandem mass spectrometry, in conjunction with MOD i and MODmap algorithm. We observed unexpected mass shifts (⌬m ‫؍‬ ؊16, ؊34, ؉64, ؉87, and ؉103 Da) at redox-active cysteine residue in cellular GAPDH purified on 2D-PAGE, in oxidized NDP kinase A, peroxiredoxin 6, and in various mitochondrial proteins. Mass differences of ؊16, ؊34, and ؉64 Da are presumed to reflect the conversion of cysteine to serine, dehydroalanine (DHA), and Cys-SO 2 -SH respectively. To determine the plausible pathways to the formation of these products, we prepared model compounds and examined the hydrolysis and hydration of thiosulfonate (Cys-S-SO 2 -Cys) either to DHA (⌬m ‫؍‬ ؊34 Da) or serine along with Cys-SO 2 -SH (⌬m ‫؍‬ ؉64 Da). We also detected acrylamide adducts of sulfenic and sulfinic acids (؉87 and ؉103 Da). These findings suggest that oxidations take place at redox-active cysteine residues in cellular proteins, with the formation of thiosulfonate, Cys-SO 2 -SH, and DHA, and conversion of cysteine to serine, in addition to sulfenic, sulfinic and sulfonic acids of reactive cysteine.

Global Analysis of Myocardial Peptides Containing Cysteines With Irreversible Sulfinic and Sulfonic Acid Post-Translational Modifications

“…Information on Cys-SO 2 H/SO 3 H PTM in complex samples has thus far been generated only by amino acid analysis (hydrolyzed lysates) (14) or two-dimensional gel electrophoresis (2-DE), where these PTM cause an acidic shift (17,18). The former provides no information on specific proteins, whereas the latter relies on the modified population being of sufficient intensity for observation and/or the availability of antibodies against a protein-of-interest.…”

Proteomic Strategies for Comprehensive Identification of Post-Translational Modifications of Cellular Proteins Including Low Abundant and Novel Modifications

“…Employing SEMSA, a sensitive mass spectrometric method for detecting low abundant protein modifications, and MOD i and DBond algorithm for searching for unknown modifications of separated protein on 2D-PAGE, we characterized the nature as well as the relative abundances of these hitherto unknown Cys modifications in cellular GAPDH purified on 2D-PAGE (Jeong et al, 2011). We found unexpected mass shifts at active site Cys www.intechopen.com residue (ΔM = -16, -34 and +64 Da) in addition to those of previously known oxidation products including sulfinic and sulfonic acids, and disulfide bonds.…”

“…Results of these studies clearly established the exact oxidation sites, oxidation species, and the levels of oxidation states. This strategy was applied for finding many low abundant modifications including phosphorylation, acetylation, glutathionylation and some novel modifications (Hwang et al, 2009;Lee et al, 2010;Jeong et al, 2011). Combination of 2D-PAGE for separating modified populations and MS/MS analysis using SEMSA, makes it possible to identify low abundant modified peptides and to raise the identified peptide coverage nearly over 90%.…”

“…Cys residues are converted to Ser during oxidative stress (Jeong et al, 2011). Mass spectrometry has been extremely useful in identifying unexpected amino acid substitutions and deletion and insertion of amino acids (Fig.…”

“…We will also provide examples of novel some low abundant cysteine modifications which we identified in cellular proteins employing our strategy Hwang et al, 2009;Jeong et al, 2011). Since it is difficult in many cases, to predict the type and position of modifications from autotranslated DNA sequences, it is highly important in the postgenomic era, to experimentally obtain this information about PTMs.…”