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 low-abundant 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 MODi and MODmap algorithm. We observed unexpected mass shifts (Δm=-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-SO2-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-SO2-Cys) either to DHA (Δm=-34 Da) or serine along with Cys-SO2-SH (Δm=+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-SO2-SH, and DHA, and conversion of cysteine to serine, in addition to sulfenic, sulfinic and sulfonic acids of reactive cysteine.
Highlights
From the ‡The Center for Cell Signaling & Drug Discovery Research, College of Pharmacy, Division of Life & Pharmaceutical Sciences, Department of Bioinspired Science, Ewha Womans University, Seoul, Korea 120-750, §Department of Chemistry, KAIST, Daejeon, Korea 305-701, ¶Department of Mechanical and Information Engineering, University of Seoul, Seoul, Korea 130-743
Cellular GAPDH was purified by immunoprecipitation using flag-antibody, and the immune-complex was separated on 2D-PAGE
We looked for less abundant modifications in Cys residues in each spot using peptide sequencing with nanoUPLC-ESI-q-TOF tandem Mass spectrometry (MS), employing selectively excluded mass screening analysis (SEMSA) for sensitive detection of low abundant PTMs [17] and searching for unknown PTMs using MODi and MODmap algorithm [19, 20]
Summary
Sulfenic acids have been recently identified in cellular proteins under relatively stable environments [5,6,7], they are unstable and are readily oxidized to sulfinic (R-SO2H) and sulfonic acids, or to disulfide by condensation with free sulfhydryl. Reversible disulfide bond (Cys-SSCys) formed by condensation of sulfenic acid with free sulfhydryl, plays a key role in signaling pathways [14]. We found unexpected mass shifts at active site Cys residue (⌬m ϭ Ϫ16, Ϫ34, and ϩ64 Da) in addition to those of previously known oxidation products including sulfinic and sulfonic acids, and disulfide bonds. Sulfenic and sulfinic acids were detected as acylamide adducts (⌬m ϭ ϩ87 and ϩ103 Da) in samples on SDS-PAGE These findings suggest that diverse Cys modifications of redox-active Cys can be generated by ROS. We are conducting further studies to characterize the biological regulation and functions of these modifications
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