Fig. 4.

Overview of a generic tag-based reductive switch-labeling method for monitoring reversible protein thiol oxidation. This approach requires several steps. During Step 1 free thiols are blocked with an alkylating reagent (typically maleimide, N-ethylmaleimide, iodoacetamide, iodoacetate or methyl methane thiosulfonate). During Step 2 the reversibly oxidized thiol is reduced with the reducing agent (dithiothreitol, ascorbate or arsenite depending on the oxidation states under investigation), followed by the labeling of the nascent thiols with labeling agent (typically biotin-maleimide). Step 4 involves separation (SDS-PAGE, HPLC) and identification of the labeled protein nascent thiols (typically by immunoblotting or MS analysis).