Contribution of new mass spectrometry methods to the structural analysis of oligonucleotides

Mass spectrometry has shown its unique potential for studying the structure of proteins. Associated with various specific techniques (H/D exchange, ion mobility, gas-phase spectroscopy, multidimensional mass analysis…), it has demonstrated to be an essential tool allowing primary structures to be analyzed and providing a lot of information about high order conformations. This work assesses the capabilities of these emerging mass spectrometry methods, and especially the gas-phase H/D exchange technique, for the structural analysis ofnucleic acids. Gas-phase H/D exchange was first used to study single stranded oligonucleotides. The exchange reactions were performed with CD3OD in the collision cell of a 9.4 T FT-ICR MS. In these experimental conditions and in integrating the experimental and theoretical results, gas-phase H/D exchange was shown to be controlled by hydrogen accessibility and not by the chemical nature of the heteroatom bearing the exchangeable hydrogen. This allowed the presence of one structure or several conformers that possess different exchange properties to be detected. Moreover, when several structures were observed, increasing the internal energy of the ions at the entrance of the H/D exchange cell gave access to a qualitative estimation of the relative height of the isomerization barrierscompared to the H/D exchange ones. Ion mobility experiments confirmed independently the H/D exchange results. Comparing the ion activation experiments for H/D exchange and for ion mobility revealed that the most compact conformer displays the fastest H/D exchange. This observation showed that H/D exchange and ion mobility provide us with complementary information because accessibility and macromolecule compactness are not univocally associated. Two other methods having independent principles of operations were sequentially combined. The fragmentation of a totally deuterated dinucleotide in exchangeable positions demonstrated the coexistence of several fragmentation channels. The latter were classified according to the involvement of non-labile or labile protons in the fragmentation process. Double resonance experiments were also performed and demonstrated the existence of consecutive fragmentation mechanisms. The involvement of labile, and therefore exchangeable protons in the fragmentation mechanism casts doubt on the use of tandem mass spectrometry to localize incorporated deuteriums in oligonucleotides. Finally, an exploratory work on the gas-phase H/D exchange of non-covalent complexes is presented.