Mass spectrometry detection and reduction of disulfide adducts between reducing agents and recombinant proteins with highly reactive cysteines.

Recombinant proteins with highly reactive thiol groups can form disulfide adducts with reducing agents commonly used in protein purification, such as beta-mercaptoethanol and dithiothreitol. These adducts can interfere with protein-protein or protein-ligand interactions.This report describes the reduction of persistent disulfide adducts between the reducing agents glutathione or beta-mercaptoethanol and the recombinant protein Cyto-MelCAM, which were detected using matrix-assisted laser desorption and ionization (MALDI) mass spectrometry.These adducts were effectively reduced using the trialkylphosphine reducing agent tris(2-carboxyethyl) phosphine hydrochloride.