Chemoproteomic Profiling of Reactive Cysteines in Response to Oxidative Stress Induced by 6‐Hydroxydopamine

Although oxidative stress is a well-established driver of neurodegeneration, it remains poorly understood as to how the global cysteine (Cys) proteome is remodeled under oxidative stress conditions. Proteins with aberrantly modified cysteines in response to oxidative stress can induce and exacerbate neurodegeneration, contributing to disorders like Alzheimer's, Parkinson's, frontotemporal dementia, and amyotrophic lateral sclerosis. In this study, we induced oxidative stress in SH-SY5Y neuronal cells by subjecting them to the neurotoxin 6-hydroxydopamine (6-OHDA). To identify proteins with altered cysteine oxidation or PTM status, we used a desthiobiotin iodoacetamide (DBIA) probe, which selectively labels cysteines with unmodified and preserved thiols. Using these unbiased chemoproteomic strategies, we identified proteins with reduced Cys reactivity to DBIA in response to 6-OHDA-induced oxidative stress. Many of these proteins are critically involved in biological processes linked to cell stress responses (e.g., mitochondrial oxidative stress and apoptosis). Furthermore, we found that two key Cys on UCHL1 (a deubiquitinase critically involved in neurodegeneration) exhibited enhanced reactivity under oxidative stress conditions. Our study defines the remodeling of the Cys proteome under 6-OHDA-induced oxidative stress conditions. Furthermore, these findings suggest potential cysteine-mediated regulatory mechanisms in response to oxidative stress, providing a valuable resource for further exploration of cysteine modifications in the context of neurodegenerative signaling.