Protein-thiol oxidation subserves multiple biological functions, from enzymatic catalysis to protein oxidative folding, protein trafficking, reactive oxygen (ROS) and nitrogen (RNS) species sensing and signaling and, more generally, protein redox regulation. Protein-thiol oxidation may also constitute a sequel of ROS and RNS toxicity. Accurate and robust methods aimed at monitoring the in vivo redox state of cysteine residues are thus warranted. To this aim, we have developed biochemical approaches that rely on trapping cysteine residues in their in vivo redox state using acidic conditions, followed by the differential labeling of reduced versus oxidized cysteine residues by thiol-specific reagents. These methods have been instrumental in the discovery of eukaryotic peroxide receptors and new ROS-scavenging enzymes and in identifying the repertoire of cytoplasmic oxidized protein thiols. Proteome-wide approaches also contributed to establish the functions of the thioredoxin and glutathione pathways in eukaryotic cytoplasmic thiol-redox control.
