Site-Specific Hydrogen/Deuterium Exchange Analysis of a Protein by Mass Spectrometry Coupled with Carboxypeptidase Digestion

Abstract

Site-specific hydrogen/deuterium (H/D) exchange was investigated using a model protein, Escherichia coli dihydrofolate reductase, by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) coupled with two-step protease digestion by pepsin and carboxypeptidase P. Carboxypeptidase, which is generally inactive under quenching conditions (0 and pH 2.9), produced some overlapping peptide sequences in the presence of 3.9 mM NaCl as a stabilizer. From di#erences in the masses of these peptides, we successfully determined the deuterium incorporation of backbone amide hydrogens of six residuesMet16 (M20 loop), Glu17 (M20 loop), Met92 (bE), Ala117 (bF-bG loop), Cys152 (bH), and Phe153 (bH)at a higher resolution using the program Isotopica, which is capable of deconvoluting a complex isotope envelope associated with the isotope distribution of deuterated peptides. The H/D exchange kinetics data obtained were highly consistent with the local structure and fluctuation of these sites as revealed by nuclear magnetic resonance (NMR) and X-ray crystallography. These results demonstrate that MALDI MS coupled with two-step digestion is a useful tool for studying the site-specific H/D exchange of proteins without the deuterium scrambling that occurs in the gas-phase collision-induced dissociation method.