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Defining the intrinsic properties of amino acids which dictate the formation of helices, the most common protein secondary structure element, is an essential part of understanding protein folding. Pauling and co-workers initially predicted helical peptide folding motifs in the absence of solvent, suggesting that in vacuo studies may potentially discern the role of solvation in protein structure. Ion mobility-mass spectrometry (IMMS) combines a gas-phase ion separation based on collision cross-section (apparent surface area) with time-of-flight MS. The result is a correlation of collision cross-section with mass-to-charge, allowing detection of multiple conformations of the same ion. Most gas-phase peptide ions assume a compact, globular state that minimizes exposure to the low dielectric environment and maximizes intramolecular charge solvation. Conversely, a small number of peptides adopt a more extended (β-sheet or α-helix) conformation and exhibit a larger than predicted collision cross-section. Collision cross-sections measured using IM-MS are correlated with theoretical models generated using simulated annealing and allow for assignment of the overall ion structural motif (e.g. helix vs. chargesolvated globule). Here, two series of model peptides having known solution-phase helical propensities, namely Ac-(AAKAA)nY-NH2 (n = 3, 4, 5, 6 and 7) and Ac-Y(AEAAKA)nF-NH2 (n = 2, 3, 4, and 5), are investigated using IM-MS. Both protonated ([M + H]+) and metalcoordinated ([M + X]+ where X = Li, Na, K, Rb or Cs) species were analyzed to better understand the interplay of forces involved in gas-phase helical structure and stability. The data are analyzed using computational methods to examine the influence of peptide length, primary sequence, and number of basic (Lys, K) and acidic (Glu, E) residues on anhydrous ion structure.

IM-MS is a 2-D technique which provides separations based on ion shape (ion-neutral collision cross-section, Ω) and mass (m/z ratio). Ion structures can be deduced from the measured collision cross-section (Ωmeas) by calculating the collision cross-sections (Ωcalc) of candidates generated by molecular dynamics (MD) and compared with the experiment results. A database of Ωs for singly-charged peptide ions is presented. Standard proteins are digested using different enzymes (trypsin, chymotrypsin and pepsin), resulting in peptides that differ in amino acid composition. The majority (63%) of the peptide ion correlates well with the globular structures, but some exhibit Ωs that are significantly larger or smaller than the average correlation. Of the peptide ions having larger Ωs, approximately 71% are derived from trypsin digestion and most of the peptide ions that have smaller Ωs are derived from pepsin digestion (90%). We use computational simulations and clustering methods to assign backbone conformations for singly-protonated ions of the model peptide (NH2-Met-Ile-Phe-Ala-Gly-Ile-Lys-COOH) formed by both MALDI and ESI and compare the structures of MIFAGIK derivatives to test the ‘sensitivity’ of the cluster analysis method. Cluster analysis suggests that [MIFAGIK + H]+ ions formed by MALDI have a predominantly turn structure even though the low energy ions prefer partial helical conformers. Although the ions formed by ESI have Ωs that are different from those formed by MALDI, the results of cluster analysis indicate that the ions backbone structures are similar. Chemical modifications (N-acetyl, methylester, as well as addition of Boc or Fmoc groups) of MIFAGIK alter the distribution of various conformers, the most dramatic changes are observed for the [M + Na]+ ion, which show a strong preference for random coil conformers owing to the strong solvation by the backbone amide groups. Ωmeas of oligodeoxynucleotides in different length have been measured in both positive and negative modes. For a given molecular weight and charge state, Ωmeas of the oligodeoxynucleotide ions are smaller than those of the peptides, indicating their different packing efficiency. A novel generalized non-Boltzman sampling MD has been utilized to investigate the gas-phase ion conformations of dGGATC based on the free energy values. Theory predicts only one low-energy conformer for the zwitterionic form of dGGATC- while dGGATC+ ions have several stable conformers in both canonical and zwitterionic form in the gas phase, in good agreement with the experiment.

En plus d’être l’intermédiaire entre l’ADN et les protéines, l’ARN est impliqué dans plusieurs processus biologiques : régulation et expression des gènes (riboswitches, ARNm et ARNt) ou encore catalyse (ribozymes). La fonction de chaque ARN est liée à sa structure et à sa dynamique de repliement. Des cations tel que le magnésium se lient à l’ARN et peuvent être essentiels au bon repliement et à la stabilité de ces structures. L’obtention de détails structuraux et thermodynamiques sur l’interaction avec le magnésium a donc une grande importance dans la compréhension de la relation structure-fonction. La première partie de ce travail a consisté en la caractérisation des équilibres de liaison entre le magnésium et des motifs d’ARN modèles, appelés « kissing complexes », par spectrométrie de masse native (SM). Grâce à la SM, il est possible de distinguer les stoechiométries de liaison du magnésium. Le travail présenté ici a permis l’élaboration d’une méthode pour quantifier chaque espèce en prenant en compte la distribution d’adduits non-spécifiques. Afin d’aller plus loin dans la localisation du magnésium, nous avons utilisé la spectrométrie de masse en tandem (SM/SM). Nous avons également étudié le comportement des complexes d’ARN en phase gazeuse en utilisant la spectrométrie de mobilité ionique (SMI), avec pour but de détecter des changements de conformation dus à la liaison de cations ou ligands. Contrairement à ce qui était anticipé, nous avons démontré que les duplexes d’ADN et ARN ainsi que les « kissing complexes » subissaient une compaction significative en phase gazeuse aux états de charge initialement obtenus par SM native, ce qui pourrait cacher l’effet des cations. Notre travail a montré comment la spectrométrie de masse peut apporter de nouvelles indications sur les stoechiométries et affinités entre ARN et cations, et discute de certaines limitations quant à l’utilisation de techniques en phase gazeuse pour explorer les structures en solution. / Besides being the molecular intermediate between DNA and proteins, RNA can have many other functions such as gene regulation (riboswitches), gene expression (mRNA and tRNA) or catalysis (ribozymes). RNA function is linked to its structure and its folding dynamics. Cations such as magnesium bind to RNA and are in some instances essential for proper folding and for stability. The need of structural and thermodynamic details about Mg2+ interactions is then of upmost importance in the study of the structurefunction relationships. The first part of our work consists in characterizing the binding equilibria between magnesium and RNA model motifs, called kissing complexes, using native mass spectrometry (MS). MS makes it possible to distinguish individual binding stoichiometries, and the present work consisted in developing a method to quantify each species, taking into account the contribution of nonspecific adducts. We also explored how tandem mass spectrometry (MS/MS) could further help localizing magnesium ions. Further, we explored the structures of RNA complexes in the gas phase using ion mobility mass spectrometry (IMMS), with the aim to detect shape changes upon cation or ligand binding. But in contrast with anticipations, we found that DNA and RNA duplexes as well as RNA kissing complexes undergo a significant compaction at charge states naturally produced by native ESI-MS, which may hide the effect of cations. Our work showcases how mass spectrometry can bring novel information on RNA-cation binding stoichiometries and affinities, but also discusses some limitations of a gas-phase method to probe solution structures.