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.