Cysteine Reactivity Profiling to Unveil Redox Regulation in Phytopathogens

Reactivity-based chemical proteomics is a powerful technology based on the use of tagged chemicals that covalently react with surface-exposed residues on proteins in native proteomes. Reactivity profiling involves the purification, identification, and quantification of labeled peptides by LC-MS/MS. Here, we have detailed a protocol for reactivity profiling of Cys residues using iodoacetamide probes, displaying >1000 reactive Cys residues in the proteome of phytopathogen Pseudomonas syringae pv. tomato DC3000 (PtoDC3000). Comparative reactivity profiling of PtoDC3000 treated with or without hydrogen peroxide (H₂O₂) identified ~200 H₂O₂-sensitive Cys residues in antioxidant enzymes, metabolic enzymes, and transcription regulators. Interestingly, half of these H₂O₂-sensitive Cys residues are more reactive in response to H₂O₂ and several proteins have multiple Cys residues with opposite reactivities in response to H₂O₂ exposure.