Simultaneous Reduction and Digestion of Proteins with Disulfide Bonds for Hydrogen/Deuterium Exchange Monitored by Mass Spectrometry

摘要

Proteolyzed peptides provide the basis for mass-analyzednhydrogen/deuterium exchange (HDX) for mapping solventnaccess to various segments of solution-phase proteins.nAspergillus saitoi protease type XIII and porcine pepsinncan generate peptides of overlapping sequences and highnsequence coverage. However, if disulfide bonds arenpresent, proteolysis can be severely limited, particularlynin the vicinity of the disulfide linkage(s). Disulfide bondsncannot be reduced before or during the H/D exchangenreaction without affecting the protein higher-order structure.nHere, we demonstrate simultaneous quench/digestionreduction following H/D exchange, for subsequentnmass analysis. Proteolysis is conducted in the presencenoftris(2-carboxyethyl)phosphinehydrochloride(TCEP· HCl)nand urea, and all other steps of the H/D exchange andnanalysis are maintained. This method yields dramaticallynincreased sequence coverage and localization of solventexposednsegments for mass-analyzed solution-phase H/Dnexchange of proteins containing disulfide bonds.