Redox proteomic analysis of H2O2 -treated Jurkat cells and effects of bicarbonate and knockout of peroxiredoxins 1 and 2.

Abstract

Oxidation of thiol proteins and redox signaling occur in cells exposed to H₂O₂ but mechanisms are unclear. We used redox proteomics to seek evidence of oxidation of specific proteins either by a mechanism involving reaction of H₂O₂ with CO₂/bicarbonate to give the more reactive peroxymonocarbonate, or via a relay involving peroxiredoxins (Prdxs). Changes in oxidation state of specific Cys-SH residues on treating Jurkat T lymphoma cells with H₂O₂ were measured by isotopically labeling reduced thiols and analysis by mass spectrometry. The effects of bicarbonate and of knocking out either Prdx1 or Prdx2 were examined. Approximately 14,000 Cys-peptides were detected, of which ∼1 % underwent 2-10 fold loss in thiol content with H₂O₂. Those showing the most oxidation were not affected by the presence of bicarbonate or knockout of either Prdx. Consistent with previous evidence that bicarbonate potentiates inactivation of glyceraldehyde-3-phosphate dehydrogenase, the GAPDH active site Cys residues were significantly more sensitive to H₂O₂ when bicarbonate was present. Several other proteins were identified as promising candidates for further investigation. Although we identified some potential protein candidates for Prdx-dependent oxidation, most of the significant differences between KO and WT cells were seen in proteins for which H₂O₂ unexpectedly increased their CysSH content over untreated cells. We conclude that facilitation of protein oxidation by bicarbonate or Prdx-mediated relays is restricted to a small number of proteins and is insufficient to explain the majority of the oxidation of the cell thiols that occured in response to H₂O₂.