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

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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°C and pH 2.9), produced some overlapping peptide sequences in the presence of 3.9 mM NaCl as a stabilizer. From differences in the masses of these peptides, we successfully determined the deuterium incorporation of backbone amide hydrogens of six residues—Met16 (M20 loop), Glu17 (M20 loop), Met92 (βE), Ala117 (βF-βG loop), Cys152 (βH), and Phe153 (βH)—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.
日本質量分析学会の論文
2007-10-01