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

Abstract

Gas-phase hydrogen/deuterium exchange of small oligonucleotides (dTG, dC 6 and C 6 ) with CD 3 0D was performed in the second hexapole of a Fourier transform ion-cyclotron resonance (FTICR) mass spectrometer. Ion activation experiments were conducted by accelerating the ions at the entrance of the H/D exchange cell under conditions promoting exclusively collisional isomerization. These experiments allowed us to assess the presence of several conformers, and to probe the height of the isomerization barrier separating these conformers. 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. 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 Address reprint requests to Miss Dorothee Balbeur, Laboratory of Mass Spectrometry, University of Liege, 3 Allee de la Chimie Bat. B6c, B-4000 Liege, Belgium. 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. Furthermore, gas-phase H/D exchange of dinucleotides has been shown to be controlled by hydrogen accessibility rather than by the chemical nature of the heteroatom bearing the exchangeable hydrogen. Studies of larger oligonucleotides have also been published. Gas-phase H/D exchange reactions were performed on DNA duplexes and on the corresponding single strands. They showed that H/D exchange was slower for the duplex than for the single strands [15, 16J. In a previous study, a quadruplex was shown to undergo very fast H/D exchange compared with its constituent monomer, an unexpected situation for such a compact and rigid structure [17J. Gas-phase H/D exchange of 5-mer phosphorothioate oligonucleotides was also examined; bimodal exchange profiles were observed, suggesting that each ion could adopt more than one gas-phase conformation [18J.The present work focuses on the bimodal H/D exchange of oligonucleotides. A simple interpretation of such a behavior is that one ...

Towards integrative structural mass spectrometry: Benefits from hybrid approaches

“…Purves and co-workers showed that each individual technique could identify only 2 conformers for the [M+2H] 2+ ion of bradykinin while 4 conformers were characterized by combining both methods. Complementary information can also be gathered by combining low-field ion mobility (either with a drift tube or a T-Wave instrument) and gas-phase HDX, as shown for oligonucleotides [117]. Finally, a recent setup consisted of a combination of low-field ion mobility and solution HDX.…”

A eole for the MS analysis of nucleic acids in the post-genomics age

“…However, this approach has been successfully employed to highlight the protein surfaces in contact with them and to study the dynamics associated with the specific interactions [163][164][165]. The implementation of HDX in the gas phase has been demonstrated for nucleotides [166,167], unstructured oligonucleotides [168], and relatively small structured constructs [169,170], but its application to the larger assemblies has yet to be reported.…”

“…The conformational study of progressively larger assemblies will be expected to take full advantage of recent exciting developments in ion mobility mass spectrometry, which has been already shown to be capable of affording direct information about nucleic acid structures in the gas phase [169,176].…”

Gas-phase hydrogen/deuterium exchange of dinucleotides and 5′-monophosphate dinucleotides in a quadrupole ion trap

“…In recent years, these studies have been augmented by extensive molecular modeling in an effort to provide further support for mechanistic and structural details of the H/D exchange reactions of small organic molecules [1–3,6,8,9,14,17,24], as well as affording incredible insight into the gas-phase conformations of biological molecules, such as proteins and DNA [11,20,25,28,29]. The gas-phase structures of mononucleotides and dinucleotides [4,5,12,13,15,23], in addition to some large oligonucleotides [7,11,20,25,27,], have been examined by H/D exchange methods in a number of studies using different deuterating reagents and types of mass spectrometers.…”

“…Gas-phase H/D exchange experiments [26,27] and ion mobility measurements of dinucleotides have also been reported [31,32]. The ion mobility studies concluded that multiple gas-phase conformations of similar energy existed and that the conformations were both nucleobase- and sequence-dependent.…”