Reactive oxygen/nitrogen species (ROS/RNS) are commonly produced during cellular metabolism, having an important role in the regulation of several pathways under physiological conditions. Nevertheless, an excessive generation of ROS/RNS combined with impaired antioxidant defenses results in oxidative or nitrosative stress, which is associated with deleterious effects, such as modification of protein residues, disruption of cellular homeostasis, and disease development. Thiol groups are particularly vulnerable to oxidation, with the modifications induced by ROS/RNS in cysteine residues leading to alteration of protein function. In fact, cysteine thiol modifications are frequently identified in both physiological and pathophysiological conditions, making their study a field of growing importance.

The identification and quantification of cysteine redox modifications can be performed using several approaches developed in the last years based on the use of thiol-specific reagents and isotope tags. Most importantly, some of those methods can preserve the physiological thiol oxidative state of the proteins/small thiols, and can differentiate the different types of cysteine reversible oxidations. The combination of these strategies with mass spectrometry enables a qualitative and quantitative characterization of the thiol modifications under different conditions.

Thus, in this methods collection, we aim at gathering the most valuable and current approaches to study redox proteomics (and small thiols), with a particular focus on the challenges and solutions associated with this type of samples, including sample preparation and quantitation strategies, MS setup, operation, and data analysis.