Fig. 3.

Biotin-switch techniques (BST) and variations for the proteome-wide identification and quantification of thiol redox modifications. A, BST involve differential thiol labeling of reduced and oxidized thiols. Free thiols are first blocked with a small-molecule thiol-reactive agent such as iodoacetamide (IA), N-ethylmaleimide (NEM), or methyl methanethiosulfonate (MMTS). Reversibly oxidized thiols are reduced with a specific reducing agent, labeled with another thiol-reactive agent with a functionalized group, such as biotin, TMT, or resin, enriched by affinity purification or resin-assisted capture, and analyzed by MS-based proteomics. B, OxICAT is used to measure the relative content of oxidized thiols in thiol proteome. Free thiols are first labeled with the light (¹²C) ICAT (red), while oxidized proteins in the same sample are subsequently reduced and labeled with the heavy (¹³C) ICAT (blue). The ICAT labeled sample is then subjected to trypsin digestion and affinity purification. The resulting peptides are detected and quantified by MS-based proteomics. The extent of thiol oxidation in any given peptide is determined by the ratios of heavy to light precursor ion signal intensity.