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

This is the authors' abstract. We don't add key points for this paper.

Oxidation of thiol proteins and redox signaling occurs in cells exposed to H 2 O 2 but mechanisms are unclear. We used redox proteomics to seek evidence of oxidation of specific proteins either by a mechanism involving reaction of H 2 O 2 with CO 2 /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 H 2 O 2 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 H 2 O 2 . 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 H 2 O 2 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 H 2 O 2 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 cell thiols that occurs in response to H 2 O 2 . • The effect of 100 μM H 2 O 2 on the reduced thiol proteome of Jurkat cells was determined. • A more than 2-fold loss in -SH was seen for ∼1% of the 14,000 Cys-peptides detected. Most of the changes including the major ones were independent of bicarbonate. • Oxidation of GAPDH and several other proteins was enhanced by bicarbonate. • The 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.