Non-Reducing Proteomics Reveals Disulfide-dependent Proteoform Remodeling Under Oxidative Stress

Abstract

Oxidative stress triggers redox-sensitive post-translational modifications, notably disulfide bond formation involving cysteine residues. However, these bonds are often overlooked in proteomics due to the routine use of reducing agents. Here, we employed (LC-MS based metabolomics and non-reducing tandem mass tag proteomics to investigate the effects of hydrogen peroxide on MDA-MB-231 cells. Metabolomic analysis revealed pathway-specific inhibition of major metabolic pathways including glycolysis, the tricarboxylic acid cycle, and nucleotide biosynthesis. Proteomic analysis using the DBond algorithm revealed extensive and isoform-specific disulfide crosslinks across more than 1000 proteins. These linkages were enriched at redox-sensitive cysteines near basic residues and displayed high isoform specificity. Our findings demonstrate that disulfide bond formation serves as a selective mechanism of redox regulation. This study highlights the utility of non-reducing proteomics in elucidating redox-controlled protein networks and structural dynamics under oxidative stress.