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

Electron detachment dissociation (EDD) pathways in oligonucleotides

Electron detachment dissociation (EDD) and electron photodetachment dissociation (EPD) are two novel fragmentation methods yielding radicals from negatively charged ions. With the goal of comparing EDD, EPD and the more traditional Collision-Induced Dissociation (CID) and Infrared Multiphoton Disscociation (IRMPD) fragmentation processes in oligonucleotides, we studied here the EDD fragmentation pathways of oligonucleotides of varying length. We chose polythymine oligonucleotides because these are the least prone to secondary structure formation, and found complete sequence coverage by EDD for up to dT20. We also found that the fragmentation pathways change with oligonucleotide length: electron detachment is a mandatory step in the fragmentation of larger sequences, while shorter oligonucleotides can also fragment via direct electronic or vibrational excitation by the electrons. This is supported by (1) the fact that continuous ejection of the charge reduced species does not totally prevent fragmentation of short oligonucleotides dT5 and dT6, (2) the fact that CID and EDD fragments are more similar for small oligonucleotides (although double resonance experiments show that they are not all issued from the same mechanisms), and (3) the fact that electron-induced dissociation (EID) of singly charged dT3 and dT4 gives similar fragments as EDD of doubly charged dT5 and dT6. Finally, the detachment efficiency as a function of the nature of the nucleobase was studied. The effect of base on electron detachment in EDD (G > T > A > C) is different than in EPD (G > A > C > T), indicating different electron loss mechanisms

Identification of Fragmentation Channels of Dinucleotides Using Deuterium Labeling

The fragmentation of the totally deuterated dinucleotide dAT− in labile positions (heteroatom-bound hydrogens) was compared for different MS/MS methods: CID, IRMPD, and EID. These experiments allowed us to affirm the coexistence of several fragmentation channels. They can be classified according to the involvement of nonlabile or labile protons in the fragmentation process. Moreover, double resonance experiments were performed in IRMPD and EID. They demonstrated the existence of consecutive fragmentation processes. The probability with which each channel is taken depends on the fragmentation technique used, i.e., the energy and the time scale of the method. The fragmentation channels that involve labile protons requiring peculiar three-dimensional structures are entropically unfavorable and enthalpically favorable. They are more observed in IRMPD and EID. 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

Conformationally Driven Gas-Phase H/D Exchange of Dinucleotide Negative Ions

Gas-phase hydrogen/deuterium exchange of six deprotonated dinucleotides with CD3OD was performed in the second hexapole of a Fourier transform ion-cyclotron resonance (FTICR) mass spectrometer. To complete these experiments, dynamic simulations were carried out to investigate the different conformations adopted by the dinucleotides. In the experimental conditions and in integrating the experimental and theoretical results, H/D exchange was shown to be controlled by hydrogen accessibility and not by the chemical nature of the heteroatom bearing the exchangeable hydrogen. A model including simultaneous H/D exchanges at the experimental time scale was used to reproduce the dinucleotide H/D exchange kinetic plots. The relay mechanism was not relevant for dinucleotides. This allowed the H/D exchange rates to be directly linked to conformations