Proteomic Profiling of Oxidative Stress Response Proteins with a Methionine Sulfoxide-Inspired Activity-Based Probe

Methionine oxidation is a key hallmark of cellular oxidative stress, which is reversed by methionine sulfoxide reductases (Msrs) as part of cellular defense mechanisms. Current tools for studying methionine oxidation and Msr function rely on sulfoxide reduction or transcriptional analysis, which are inadequate for monitoring enzyme activity under persistent oxidative stress. Moreover, no activity-based protein profiling (ABPP) tools have been reported for investigating the functional state of Msrs in cells. Here, we present SO-acetylene, a methionine sulfoxide-inspired, cysteine-reactive probe for profiling Msrs and other proteins involved in oxidative stress responses. This substrate-inspired probe preferentially labels catalytic cysteines of Msrs, serving as an activity-based probe. The application of SO-acetylene to <i>Escherichia coli</i> under hypochlorite stress resulted in the labeling of multiple oxidative stress-related proteins, revealing distinct activity patterns among Msrs that diverge from transcriptional regulation. Furthermore, comparative labeling experiments with the conventional probe iodoacetamide revealed that SO-acetylene provides complementary coverage of cysteine reactivity, efficiently capturing the active-site cysteine of a DJ-1 superfamily glyoxalase, which is poorly labeled by iodoacetamide-alkyne.