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

• Published in: Free radical biology & medicine 2024-11, Vol.227, p.221-232
• Main Authors: Pace, Paul E., Fu, Ling, Hampton, Mark B., Winterbourn, Christine C.
• Format: Article
• Language: English
• Subjects: Hydrogen peroxide; Peroxiredoxin; Peroxymonocarbonate; Redox relay; Thiol proteome
• ISSN: 0891-5849; 1873-4596; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2024.10.314

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. [Display omitted] •The effect of 100 μM H2O2 (20 nM/106 cells) on the reduced thiol proteome of Jurkat cells was determined.•More than 2-fold loss in -SH was seen for ∼1 % of 14,000 Cys-peptides.•Most changes were independent of bicarbonate.•Oxidation of GAPDH and several other proteins was enhanced by bicarbonate.•Proteins showing the most oxidation were not affected by knockout of Prdx1 or Prdx2.•No compelling evidence for new Prdx-mediated redox relays was revealed.