Gas-phase hydrogen/deuterium exchange of 5′- and 3′-mononucleotides in a quadrupole ion trap: Exploring the role of conformation and system energy

Abstract

Gas-phase hydrogen/deuterium (H/D) exchange reactions for deprotonated 2=-deoxy-5=-monophosphate and 2=-deoxy-3=-monophosphate nucleotides with D 2 O were performed in a quadrupole ion trap mass spectrometer. To augment these experiments, molecular modeling was also conducted to identify likely deprotonation sites and potential gas-phase conformations of the anions. A majority of the 5=-monophosphates exchanged extensively with several of the compounds completely incorporating deuterium in place of their labile hydrogen atoms. In contrast, most of the 3=-monophosphate isomers exchanged relatively few hydrogen atoms, even though the rate of the first two exchanges was greater than observed for the 5=-monophosphates. Mononucleotides that failed to incorporate more than two deuterium atoms under default reaction conditions were often found to exchange more extensively when reactions were performed under higher energy conditions. Integration of the experimental and theoretical results supports the use of a relay exchange mechanism and suggests that the exchange behavior depends highly on the identity and orientation of the nucleobase and the position and flexibility of the deprotonated phosphate moiety. These observations also highlight the importance of the distance between the various participating groups in addition to their gas-phase acidity and basicity. (J Am Soc Mass Spectrom 2007, 18, 724 -736)

Detection of oligonucleotide gas-phase conformers: H/D exchange and ion mobility as complementary techniques

“…Although it is intuitively surprising, it is conceivable that a compact structure possesses exchangeable hydrogens with high H/D exchange rate constants. To account for these high rate constants, direct accessibility of hydrogens or more probably various intramolecular "relay" mechanisms (not to be confused with the relay mechanism that is mentioned in the mononucleotides studies [7][8][9][10][11]), which can bring deuteriums into the compact structure, and/or H/D exchange barriers changes upon folding can be evoked. A detailed structural study of C 6 2Ϫ searching for different stable struc- tures and possibly comparing the theoretical and experimental cross sections would be very relevant.…”

“…Hydrogen/deuterium (H/D) exchange combined with mass spectrometry has become an efficient tool for obtaining conformational information about isolated biomolecules in the gas phase [1-18J. The rate of gasphase H/D exchange has been shown to be a function of the reagent used for exchange (nature and concentration), the charge state of the biomolecule, the gasphase basicity/acidity of exchangeable sites, and the internal structure of the biomolecular ions [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18]. Probing the number of exchanged hydrogens and the H/D exchange kinetics makes it possible to distinguish ion populations and to elucidate their structural and thermochemical features.…”

“…E-mail: dbalbeur@ulg.ac.be that the extent and rate of exchange depend on the nucleobase (nature, space orientation, and gas-phase acidity of the exchangeable hydrogen), and on the position (3' or 5') and flexibility of the terminal phosphate group [7-11J. Moreover, these studies have been in favor of the relay mechanism [7][8][9][10][11]. The latter was shown not to be relevant for dinucleotides [14J.…”

“…Probing the number of exchanged hydrogens and the H/D exchange kinetics makes it possible to distinguish ion populations and to elucidate their structural and thermochemical features. Gas-phase H/D exchange has been applied extensively for structural studies of proteins and peptides [1-6J and is now also most commonly used for structural characterizations of oligonucleotides [7][8][9][10][11][12][13][14][15][16][17][18]. Mononucleotides studies have shown that the extent and rate of exchange depend on the nucleobase (nature, space orientation, and gas-phase acidity of the exchangeable hydrogen), and on the position (3' or 5') and flexibility of the terminal phosphate group [7-11J.…”

Gas-Phase Hydrogen-Deuterium Exchange Labeling of Select Peptide Ion Conformer Types: a Per-Residue Kinetics Analysis

“…Other experiments detailed HDX characterization of a variety of protein ions demonstrating the presence of multiple conformations as well as revealing factors that affect HDX of specific ions such as conformational rigidity [7]. Gas-phase HDX-MS experiments also extended to the characterization of other species, including small organic molecules [11,12], nucleotides [13][14][15][16], and peptides [17,18].…”