Electrospray ionization mass spectrometry (ESI-MS) has been applied to the realtime study of homogeneous organometallic reactions. ESI-MS as a soft ionization technique is amenable to fragile organometallic complexes, and as a fast and sensitive technique is ideal for detecting low concentration intermediates within reactions. Pressurized sample infusion (PSI) was used for continuous sample infusion into the mass spectrometer, granting the air-free conditions necessary for these reactions to be successful, and resulting in reaction profile data that contains information about the dynamics of speciation of the catalyst. Collision induced dissociation (CID) was used to probe the binding affinities of various bisphosphine ligands as well as in characterizing intermediates in reactions. PSI ESI-MS was applied to the hydroboration reaction of the alkene tert-butylethene using the amine-borane H3B⋅NMe3 catalyzed by [Rh(xantphos)]+ fragments to show how the reaction progresses from substrates to products. PSI ESI-MS was also applied to the hydrogenation of a charge-tagged alkyne [Ph3P(CH2)4C2H]+[PF6]-, catalyzed by a cationic rhodium complex [Rh(PcPr3)2(η6-FPh)]+[B{3,5-(CF3)2C6H3}4]– (PcPr3 = triscyclopropylphosphine, FPh = fluorobenzene). This work demonstrated the use of ESI-MS in conjunction with NMR, kinetic isotope effects and numerical modeling for determining a mechanism of reaction. The hydroacylation reaction of a β–S substituted aldehyde and an alkyne catalyzed by [Rh(PiPr2NMePiPr2)(η6-FPh)]+[B{3,5-(CF3)2C6H3}4]– (PiPr2 = diisopropylphosphine) was studied by PSI ESI-MS while employing charged tags, allowing for observation of reaction progress and some key intermediates. A new concept for mechanistic analysis has been developed: coupling of an orthogonal spectroscopic technique with PSI ESI-MS. This new method was applied to the same hydroacylation reaction studied with charged tags. The use of IR in conjunction with ESI-MS led to rate information about the overall reaction along with dynamic information about catalytic speciation. Coupling of these techniques allows for detection over many magnitudes of concentration. / Graduate

Ce travail de thèse porte sur le développement de méthodes de spectrométrie de masse (MS) structurale pour la caractérisation de systèmes protéiques complexes, souvent réfractaires aux approches biophysiques classiques. Dans ce contexte, les développements entrepris furent notamment focalisés sur la caractérisation de complexes impliqués dans la biogénèse des ribosomes et dans la régulation transcriptionnelle, fonctions cellulaires essentielles pouvant être liées à de nombreuses pathologies humaines dont certains cancers. Ainsi, les approches par MS native, pontage chimique et d’HDX-MS ont permis de renseigner sur la connectivité, les proximités spatiales ou encore la dynamique conformationnelle retrouvées au sein des complexes étudiés. Parmi ces techniques, l’HDX-MS permet une approche comparative basée sur les mesures d’incorporations en deutérium renseignant sur la dynamique conformationnelle d’une protéine sous différents états. Aussi, la combinaison d’approches de MS structurale a permis d’approfondir la caractérisation des systèmes complexes étudiés, démontrant ainsi l’intérêt d’une approche intégrative dans ce contexte. / This PhD thesis focuses on developing methods in structural mass spectrometry (MS) to characterize complex protein systems, given their size and their heterogeneity, frequently inaccessible by classical biophysic approaches. In this context, methodological developments have particularly focused on the characterization of protein complexes involved in ribosomes biogenesis and transcriptional regulation. These fundamental cellular processes are related to numerous diseases such as cancers and genetic diseases. Thus native MS, crosslink, and hydrogen/deuterium exchange coupled to MS (HDX-MS) allowed gaining insights about the stoechiometry, spatial proximities and conformational dynamics of studied systems. Among these approaches, HDX-MS enables a comparative approach based on deuterium incorporation measurements giving information about the conformational dynamics of labeled proteins in various experimental conditions. Finally, the combination of structural approaches enables to deeply characterize complex protein systems, highlighting the advantages of an integrative approach in this context.