The protein cysteine residue is one of the amino acids most susceptible to oxidative modifications, frequently caused by oxidative stress. Several applications have enabled cysteine-targeted proteomics analysis with simultaneous detection and quantitation. In the current study, we employed a quantitative approach using a set of iodoacetyl-based cysteine reactive isobaric tags (iodoTMT) and evaluated the transient cellular oxidation ratio of free and reversibly modified cysteine thiols under DTT and hydrogen peroxide (H2O2) treatments. DTT treatment (1 mM for 5 min) reduced most cysteine thiols, irrespective of their cellular localizations. It also caused some unique oxidative shifts, including for peroxiredoxin 2 (PRDX2) and thioredoxin (TXN), proteins reportedly affected by cellular reactive oxygen species production. Modest H2O2 treatment (50 µM for 5 min) did not cause global oxidations but, instead, had apparently reductive effects. Moreover, with H2O2, significant oxidative shifts were observed only in redox active proteins, like PRDX2, peroxiredoxin 1 (PRDX1), TXN and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). We also employed bifunctional electrophile cross-linker based interaction analysis, to explore the interaction partner of redox active proteins under these stress oonditions. Overall, our quantitative data illustrated both H2O2- and reduction-mediated cellular responses, whereby, while redox homeostasis is maintained, highly reactive thiols can potentiate the specific, rapid cellular signaling to counteract acute redox stress.

Proteomics-based screening and identification method for redox sensitivities of global cellular cysteine residues

253rd National Meeting (2017)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.