Data-Independent Acquisition (DIA)-Based Label-Free Redox Proteomics (DIALRP) Identifies Prominent Cysteine Oxidations in Translation Machinery in Prostate Cancer Cells Under Oxidative Stress

Oxidative stress is a key factor in numerous physiological
and
pathological processes, including aging, cancer, and neurodegenerative
diseases. Protein cysteine residues are particularly susceptible to
oxidative stress-induced modifications that can alter their structure
and function, thereby affecting intracellular signaling pathways.
In this study, we performed a data-independent acquisition mass spectrometry
(DIA-MS)-based label-free redox proteomics method, termed DIALRP,
to comprehensively analyze cysteine oxidative modifications in the
prostate cancer cell line DU145 under oxidative stress induced by
menadione (MND). Of 10,821 cysteine-containing peptides identified,
we successfully quantified the redox changes in 3665 peptides. We
also observed that 1407 peptides were significantly oxidized in response
to MND treatment. Gene ontology analysis revealed that a group of
translation-related molecules was most enriched among highly MND-sensitive
cysteine-containing proteins. Notably, our data demonstrated that
MND-induced oxidative stress inhibits EIF2, EIF6, and EEF2 complex
formation, suggesting that these complex inhibitions become functional
factors for a dramatic reduction in translation activity. Our results
show that DIALRP is utilized as a robust and cost-effective approach
for investigating redox-regulated cellular processes. Moreover, these
findings provide significant insights into translation regulation
under oxidative stress and provide a valuable framework for future
studies on redox-mediated cellular processes.