2244
J. Phys. Chem. B 2001, 105, 2244-2249
Assignment of Gas-Phase Dipeptide Amide Hydrogen Exchange Rate Constants by Site-Specific Substitution: GlyGly Fei He and Alan G. Marshall*,† National High Magnetic Field Laboratory, Florida State UniVersity, Tallahassee, Florida 32310
Michael A. Freitas The Ohio State UniVersity, Columbus, Ohio 43210 ReceiVed: September 27, 2000; In Final Form: January 9, 2001
Amide hydrogen deuterium exchange is widely used to probe conformations of gas-phase biomacromolecular ions. Meaningful interpretation of such experiments depends on relating the experimentally determined H/D exchange rate constants to specific exchangeable hydrogen sites on the molecule. Here, we present the first definitive assignment of H/D exchange rate constants for the N-terminal ammonium, amide hydrogen, and carboxyl hydrogens in diglycine, based on comparison of site-specific gas-phase H/D exchange rate constants for diglycine (GlyGly) and its selectively methylated homologues (GlySar, GlyGly-OMe, and GlySar-OMe). The present method may be extended to identification of site-specific H/D exchange rate constants for other dipeptides, to provide insight into H/D exchange reaction mechanisms.
Introduction After the advent of “soft” ionization techniques, such as electrospray ionization (ESI)1,2 and matrix-assisted laser desorption,3 the structure of gas-phase biomoleules received considerable attention. It is now possible to study protein folding and unfolding in the absence of solvent, and as a function of the charge state of the protein. Although a protein’s ability to fold back to its biological active conformer from a denatured/ unfolded state has been demonstrated in solution,4 the role that solvent plays in the process is not fully understood. It has been demonstrated recently that an enzyme can retain its biological activity in the gas phase (unsolvated)5 or in an organic solvent.6 These results suggest that a protein upon desolvation may retain some degree of its native solution-phase structure. However, no technique presently exists for direct determination of the three-dimensional gas-phase structure of a protein. Various mass spectrometry methods including ion mobility,7-9 proton transfer,10 hydrogen/deuterium exchange,11-15 blackbody infrared radiative dissociation,16,17 and infrared multiphoton dissociation (IRMPD),18 are being used to investigate gas-phase protein structures. H/D exchange has been combined traditionally with NMR19-21 or mass spectrometry22-24 to investigate protein solution-phase structural dynamics. The hydrogen/ deuterium exchange technique has also been adapted to study gas-phase peptide and protein conformational changes.11-14 Gasphase H/D exchange has been a powerful method capable of revealing multiple gas-phase protein conformations and gasphase zwitterions of the peptide, bradykinin.25 For solution-phase H/D exchange, the amide proton NMR signals are readily assigned according to their chemical shifts (and their much slower rate of H/D exchange than side-chain or terminal hydrogens). Thus, it has proved possible to assign and measure the H/D exchange rate constants for the amide hydrogen in each of the dipeptides formed from the 20 most
common amino acids.26 In the gas phase, on the other hand, the H/D exchange rate constants for hydrogens at different sites in a dipeptide are much more similar in magnitude, and their correct assignment has proved to be much more difficult. Many reports have been published on gas-phase H/D exchange of dipeptides,27-31 and attempts have been made to differentiate H/D exchange from different sites on peptides.27,32,33 For example, Campbell et al.29 reported that, for glycine oligomers reacted with D2O, the overall exchange rate for the oligomer slows down as the size of the oligomer increases. Complexation of peptides to crown ether or saccharides also has been used to investigate site specificity of H/D exchange.34,35 Green and Lebrilla31 showed that the single amino acid, glycine, reacted with CH3OD has one fast and three slow exchange sites, whereas diglycine reacted with CH3OD has three fast, one medium, and one slow exchange site, with relative rate constants, 8.4:8.4: 8.4:3.4:1.0. Although it is likely that the three equivalent sites are three N-terminal ammonium hydrogens (including the proton that brings charge to the molecule), it was not possible to assign the two remaining rate constants to the amide vs carboxylic hydrogens. In fact, there is still no reported definitive assignment of amide H/D exchange rate constants for gas-phase peptides. Here, we offer a method to determine directly the site-specific rate for amide hydrogen/deuterium exchange in a gas-phase dipeptide. Methylation on different sites of a diglycine peptide serves to encode gas-phase H/D exchange kinetics observed by mass spectrometry. We are then able to assign and extract sitespecific gas-phase H/D exchange rate constants.36 Deuterated water and methanol were reacted with the target peptides, and site-specific rate constants determined with excellent precision (
