A cryogenic (325-80 K) ion mobility-mass spectrometer was designed and constructed in order to improve the analytical figures-of-merit for the chemical analysis of small mass analytes using ion mobility-mass spectrometry. The instrument incorporates an electron ionization source, a quadrupole mass spectrometer, a uniform field drift cell spectrometer encased in a cryogenic envelope, and an orthogonal geometry time-of-flight mass spectrometer. The analytical benefits of low temperature ion mobility are discussed in terms of enhanced separation ability, ion selectivity and sensitivity. The distinction between resolving power and resolution for ion mobility is also discussed. Detailed experimental designs and rationales are provided for each instrument component. Tuning and calibration data and methods are also provided for the technique. Proof-of-concept experiments for an array of analytes including rare gases (argon, krypton, xenon), hydrocarbons (acetone, ethylene glycol, methanol), and halides (carbon tetrachloride) are provided in order to demonstrate the advantages and limitations of the instrument for obtaining analytically useful information. Trendline partitioning of small analyte ions based on chemical composition is demonstrated as a novel chemical analysis method. The utility of mobility-mass analysis for mass selected ions is also demonstrated, particularly for probing the ion chemistry which occurs in the drift tube for small mass ions. As a final demonstration of the separation abilities of the instrument, the electronic states of chromium and titanium (ground and excited) are separated with low temperature. The transition metal electronic state separations demonstrated here are at the highest resolution ever obtained for ion mobility methods. The electronic conformational mass isomers of methanol (conventional and distonic) are also partially separated at low temperature. Various drift gases (helium, neon, and argon) are explored for the methanol system in order to probe stronger ion-neutral interaction potentials and effectuate higher resolution separations of the two isomeric ions. Finally, two versatile ion source designs and a method for axially focusing ions at low pressure (1-10 torr) using electrostatic fields is presented along with some preliminary work on the ion sources.

Identification of trace unknown analytes in complex samples remains a significant challenge for analytical chemistry. Mass spectrometry (MS) and analytical separations techniques can now be used to develop and support a new analytical strategy called non-target analysis which aims to provide comprehensive identification and quantification of all detectable chemical species in a complex sample. This thesis addresses challenges currently limiting the utility of this non-target approach by developing analytical methods for acquiring MS data suitable for identification of trace unknowns and investigating current tools available for unknown identification from MS spectral data. Liquid chromatography (LC) - MS, a widely used technique in trace analysis, was used to develop an analytical method capable of simultaneously acquiring high resolution MS and tandem mass spectrometry (MS/MS) data for hundreds of metabolites in urine. An emerging separation technique called high field asymmetric waveform ion mobility spectrometry (FAIMS) was also investigated, as an alternative to LC, for the identification of non-target analytes in urine. Modifications were carried out to the FAIMS-MS source interface allowing for transmission of small metabolite ions from FAIMS to MS. The challenge of direct electrospray (ESI) in urine analysis using ESI-FAIMS-MS was addressed by using sample dilution and extending MS data acquisition time using FAIMS. This allowed for higher quality MS data to be acquired for low abundance urinary metabolites than was possible by LC-MS and the complete elimination of ionization suppression in dilute urine samples. Insight gained into ESI suppression in complex samples allowed for two methods of semi-quantification to be proposed for non-target analytes in complex samples without using unavailable chemical standards. To address the challenge of unknown identification, faced throughout this thesis, an integrated approach was implemented to identify metabolites based only on spectral data without the usual requirement of availability of chemical standards. This approach combined spectral libraries, literature reports on ion chemistry and de novo identification based on gas phase ion chemistry with a detailed fragmentation study on nucleic acid bases, notably protonated uracil. Together, the instrumental methods and approaches to data analysis described allowed for the identification of 110 abundant chemical species detected in urine. / Natural Sciences and Engineering Research Council of Canada, Ontario Ministry of Training, Colleges and Universities, Canadian Foundation for Innovation

Ion mobility spectrometry (IMS) separates ions on the basis of ion-neutral collision cross-sections (CCS, [omega]), which are determined by the geometry or conformation of the ions. The size-based IM separation can be extended to distinguish conformers that have different shapes in cases where shape differences influence the accessible surface area of the molecule. In recent years, IM has rapidly evolved as a structural characterization technique, which has applied on various structural biology problems. In this work, IMS is combined with molecular dynamics simulation (MDS), specially the integrated tempering sampling molecular dynamics simulation (ITS-MDS) to explore the gas-phase conformation space of two molecular systems (i) protonated tryptophan zipper 1 (trpzip1) ions and its six derivatives (ii) alkali metal ion (Na, K and Cs) adducts of gramicidin A (GA). The structural distributions obtained from ITS-MDS are compared well with results obtained from matrix-assisted laser desorption ionization-ion mobility-mass spectrometry (MALDI-IM-MS) for trpzip 1 series and electrospray ionization-ion mobility-mass spectrometry (ESI-IM-MS) for alkali metal ion adducts of GA. Furthermore, the solvent dependence on conformational preferences of the GA dimer is investigated using a combination of mass spectrometry techniques, viz. ESI-IM-MS and hydrogen/deuterium exchange (HDX)-MS, and MDS. The IM experiments reveal three distinct gramicidin A species, detected as the sodium ion adduct ions, [2GA + 2Na]²⁺, and the equilibrium abundances of the dimer ions varies with solvent polarity. The solution phase conformations are assigned as the parallel and anti-parallel [beta]-helix dimer, and the anti-parallel dimer is the preferred conformation in non-polar organic solvent. The calculated CCS profiles by ITS-MDS agree very well with the experimentally measured CCS profiles, which underscore the utility of the method for determining candidate structures as well as the relative abundances of the candidate structures. The benefit of combining ion mobility measurements with solution-phase H/D exchange is allowing identifications and detail analysis of the solution-phase subgroup conformations, which cannot be uncovered by one method alone.

Ce travail de thèse a été focalisé sur le développement d’approches MS et IM-MS supramoléculaires pour la caractérisation fine des interactions protéine/ligand et pour l’analyse de mélanges protéiques complexes. La maîtrise des instruments de MS supramoléculaire ainsi que les optimisations instrumentales et méthodologiques réalisées ont permis d’étendre le potentiel des approches MS et IM-MS pour la caractérisation d’assemblages moléculaires particulièrement complexes. Nous avons ainsi pu suivre la cinétique de formation de complexes protéine/ligand ainsi que les changements conformationnels qui y sont associés, montrant l’intérêt du couplage IM-MS en recherche pharmaceutique. De plus, ce travail a porté sur l’étude de complexes de très hauts poids moléculaires et l’évaluation de l’IM-MS pour obtenir des informations structurales sur ces complexes. Nous avons ainsi permis de repousser certaines limites de la MS et de placer cette technique au cœur des études de biologie structurale. / The aim of this thesis was the development of different supramolecular approaches, like MS and IM-MS, to characterize precisely protein/ligand interaction and to analyze complex mixtures of proteins. Understanding of supramolecular MS instruments and instrumental and methodological optimizations were allowed the development of MS and IM-MS to characterize very high mass supramolecular assembly. Thus, we were able to follow by kinetic the formation of protein/ligand interaction as well as associated conformational modifications, showing the interest of IM-MS coupling in pharmaceutical research. Furthermore, this work deals with the study of high mass complexes and assessment of IM-MS to obtain structural information on these complexes. As a consequence, we have pushed away some limits of MS allowing the use of this technique in structural biology.

Made available in DSpace on 2014-10-09T12:50:22Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:58:51Z (GMT). No. of bitstreams: 1 10890.pdf: 10562798 bytes, checksum: 063bd7e321751780a6d96cafecfe45bc (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP / FAPESP:01/06837-2

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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.

Ce travail de thèse porte sur l’analyse conformationelle de biomolécules en phase gazeuse. L’étude d’objets complexes en phase gazeuse permet de connaître les facteurs intrinsèques stabilisant leur structure. La première étape de mes travaux a été le couplage entre un appareil de mobilité ionique (IMS) et un appareil de spectrométrie de masse (MS). Nous avons simulé (SimIon) et développé différentes optiques ioniques fonctionnant à haute pression (entonnoirs à ions, piège ionique). Ces modifications expérimentales nous ont permis de commencer des études conformationnelles de biomolécules en phase gazeuse.Les premières expériences avaient pour objectif d’observer les facteurs stabilisants une structure secondaire sur des séries de peptides analogues. La première étude a été réalisée sur des séries de polyalalanines et de polyglycines de formules Arg(Ala)4XxxAla4Lys et Arg(Gly)4Xxx(Gly)4Lys, où Xxx est l’un des 20 acides aminés naturels. Nous nous sommes intéressés à l’influence de l’acide aminé central sur la conformation globale. Puis nous avons observés la stabilité de l’hélice de la partie transmembranaire de la protéine M2 du virus de la grippe A et de différents mutants. Ces études ont permit de montrer l’importance de la solvatation des charges en phase gazeuse.Enfin nous avons initié une étude sur le repliement des protéines en utilisant une protéine modèle, le lysozyme. Nous nous sommes plus particulièrement intéressés aux mécanismes de repliements en fonction du degré d’oxydation de cette dernière / This work deals with the conformational studies of biomolecules in gas phase. The gas phase allows observing the intrinsic factors that stabilize the structure.The first step of this work consisted in coupling an ion mobility tube and a mass spectrometer (IM-MS). Especially, ion optics working at high pressure had to be developed (ion funnels, cylindrical ion trap). These experimental modifications allowed us to realising conformational studies in vacuo.This work is build around two major axes. The first one deals with the understanding of the factors that stabilize the secondary structure in gas phase. We have studied series of peptides with similar sequences. We studied a series of polyalanines and polyglycines who have the following formula Arg(Ala)4XxxAla4Lys et Arg(Gly)4Xxx(Gly)4Lys, where Xxx is one of the 20 natural amino acids. We are looking the influence of central amino acid on the global structure of peptides. After that we have observed the stability of the helix of the transmembran domain of the M2 protein of virus Influenza A and some mutants. These studies allow showing the importance of the charge solvatation in the gas phase.Finally, we are initiated a study on the refolding of proteins and more particularly on a model protein: the Lysozyme. We are studied the refolding in function of oxide degrees

Cette thèse porte sur le développement d'un appareil couplant la spectrométrie de masse avec la spectrométrie de mobilité ionique et la spectroscopie laser, dans le but d'effectuer des analyses conformationnelles sur des édifices biomoléculaires. La construction, la mise au point et l'optimisation de l'appareil font l'objet de la première partie de ce mémoire. En particulier, il s'est agi de déterminer les caractéristiques de l'appareil (résolution, fréquence de fonctionnement…) par une série de tests sur des systèmes modèles. Dans un deuxième temps, nous avons effectué des mesures de spectroscopie d'action sur des conformères sélectionnés en mobilité. Nous avons étudié la relation entre la conformation et les propriétés optiques d'un système en mesurant le photo-détachement d'électron de divers conformères sélectionnés. Dans le cadre de la spectroscopie d'action, nous avons utilisé les possibilités nouvelles offertes par l'appareil pour réaliser des expériences de photo-isomérisation cis-trans sur des complexes non-covalents. Sur cet exemple, nous avons montré l'intérêt de cet appareil pour mesurer des spectres d'action de photo-isomérisation. Et enfin, nous avons montré la possibilité de réaliser des mesures de spectroscopie d'action basée sur le Transfert d'Energie par Résonance de Förster (FRET), en phase gazeuse, résolu en conformation / This thesis deals with the development of a new instrument coupling mass spectrometry, ion mobility spectrometry and laser spectroscopy. The aim is to perform structural analysis on biomolecular systems.The first part of this thesis focuses on the construction, the development and the optimization of the set-up. The main point was to determine the features of the set-up (resolution, working frequency) by series of tests with model systems.In a second phase, we did conformer resolved action spectroscopy. We studied the relation between the conformation and the optical properties of one system by measuring photo- electron detachment on different selected conformers. In the framework of action spectroscopy we used the new capacity of the set-up to perform cis-trans photo-isomerization on non-covalent complexes. We showed with this example interest of the use of this instrument to measure photo-isomerization action spectra. We finally showed the possibility to perform conformer resolved action spectroscopy measurements based on Förster Resonance Energy Transfer (FRET) in the gas phase

Ce travail de thèse porte sur le développement de méthodes en spectrométrie de masse (MS) et mobilité ionique (IM-MS) supramoléculaires pour la caractérisation fine de complexes protéine-ligand et d’assemblages protéiques hétérogènes de hauts poids moléculaires. L’optimisation instrumentale apportée à l’étude de ces systèmes, permet d’étendre le potentiel de ces deux approches en biologie structurale. Le criblage de complexes protéine-ligand permet ici une détermination de leurs propriétés d’interaction et la mise en évidence de subtils changements de conformation induits, pouvant être suivis au cours du temps. L’application de ce couplage à l’analyse de complexes multi-protéiques, réfractaires aux techniques conventionnelles, donne accès à la topologie de ces assemblages, facilitant la proposition de modèles structuraux. Enfin, l’apport récent de la haute résolution en MS native est ici illustré à travers l’étude de protéines complexes et hétérogènes : les anticorps thérapeutiques et leurs conjugués. Ces développements permettent de repousser certaines limites en MS native et IM-MS native, élargissant leurs perspectives d’application dans la recherche et l’industrie pharmaceutique. / This PhD thesis aims at developing methods in native mass spectrometry (MS) combined with ion mobility (IM-MS) to characterize protein-ligand complexes and large protein assemblies. Fine-tuning of instrumental settings allowed expanding the scope of these approaches in structural biology. Real-time monitoring of protein-ligand complexes by native MS and IM-MS enabled to screen their binding properties while depicting subtle conformational changes induced upon binding. Applying these methods to refractory multi-protein complexes provided insights about their topology, making structural modeling easier. Finally, benefits from high-resolution native MS were highlighted through the characterization of heterogeneous systems, including monoclonal antibodies and their drug conjugates. Here, these developments enable to push some technical limits one step forward, increasing the potential of native MS and IM-MS both in academic research and pharmaceutical industry.