The utility of 193 nm ultraviolet photodissociation (UVPD) and negative electron transfer dissociation (NETD) for the characterization of peptide anions was systematically evaluated. UVPD outperformed NETD in nearly all metrics; however, both methods provided complementary information to traditional collision induced dissociation (CID) of peptide cations in high throughput analyses. In order to enhance the performance of NETD, activated ion negative electron transfer dissociation (AI-NETD) methods were developed and characterized. The use of low-level infrared photoactivation or collisional activation during the NETD reaction period significantly improved peptide anion sequencing capabilities compared to NETD alone. Tyrosine deprotonation was shown to yield preferential electron detachment upon NETD or UVPD, resulting in N - C[alpha] bond cleavage N-terminal to the tyrosine residue. LC-MS/MS analysis of a tryptic digest of BSA demonstrated that these cleavages were regularly observed under high pH conditions. Transmission mode desorption electrospray ionization (TM-DESI) was coupled with 193 nm UVPD and CID for the rapid analysis and identification of protein digests. Comparative results are presented for TM-DESI-MS/CID and TM-DESI-MS/UVPD analyses of five proteolyzed model proteins. In some cases TM-DESI/UVPD outperformed TM-DESI-MS/CID due to the production of an extensive array of sequence ions and the ability to detect low m/z product ions. 193 nm UVPD was implemented in an Orbitrap mass spectrometer for characterization of intact proteins. Near-complete fragmentation of proteins up to 29 kDa was achieved. The high-energy activation afforded by UVPD exhibited far less precursor ion charge state dependence than conventional methods, and the viability of 193 nm UVPD for high throughput top-down proteomics analyses was demonstrated for the less 30 kDa protein from a fractionated yeast cell lysate. The use of helium instead of nitrogen as the C-trap and HCD cell bath gas and trapping ions in the HCD cell prior to high resolution mass analysis significantly reduced the signal decay rate for large protein ions. As a result, monoclonal IgG1 antibody was isotopically resolved and mass accurately determined. A new high mass record for which accurate mass and isotopic resolution has been achieved (148,706.3391 Da ± 3.1 ppm) was established. / text

La spectrométrie de masse est devenue un outil indispensable pour la recherche en protéomique, notamment grâce au développement récent de nouveaux spectromètres de masse comme l’Orbitrap et de nouvelles méthodes de dissociation. La stratégie « bottom-up » (analyse des mélanges de peptides protéolytiques) est la plus utilisée par son efficacement et sa simplicité par rapport à la stratégie top-down (analyse des peptides plus longs ou des protéines intactes), mais cette dernière permet une caractérisation plus complète des isoformes de protéines et des modifications post-traductionnelles.Les méthodes de dissociation utilisant des photons, comme la photodissociation dans le domaine ultra-violet (UVPD) et la dissociation multiphotonique infrarouge (IRMPD), ont reçu une grande attention comme approches alternatives aux méthodes de dissociation par collision. L'absorption du photon UV à haute énergie peut être « diluée » sur l'ensemble du peptide ou de la protéine et provoque une fragmentation étendue du squelette peptidique (liaisons C-C), tandis que les photons IR à faible énergie augmentent progressivement l'énergie interne et dissocient préférentiellement les liaisons amide (C-N) les plus labiles.Cette thèse est centrée sur le développement de méthodes et les applications pour une caractérisation structurale de biomolécules par des méthodes d'activation utilisant des photons. L'intérêt de combiner des photons infrarouges à faible énergie et des photons UV à haute énergie dans un spectromètre de masse Orbitrap, pour la caractérisation de petites protéines, a été évalué. En outre, la dissociation infrarouge multiphotonique a été implémentée dans un piège à ions électrostatique afin d’étendre les méthodes de fragmentation aux macromolécules de très haut poids moléculaires dans le domaine mégadalton. L'une des principales avancées de cette thèse a été d'adapter ces méthodes de spectrométrie de masse aux objets biomoléculaires, allant des petits peptides (dans la gamme de masse de kilodalton) à des fibres de protéines entières (dans la gamme de masse de mégadalton) / The structural characterization of proteins often required them to be fragmented into small units containing only few amino acids. In bottom-up approach, proteins are cleaved into small peptides by enzyme then these peptides are subjected to further fragmentation in a collision cell of a tandem mass spectrometer. However, in top-down approach, proteins can directly be dissociated (without enzyme) into small fragments by collision, electron and photon-driven dissociations. Photon-based activation methods including ultraviolet photodissociation (UVPD) and infrared multiphoton dissociation (IRMPD) have received great attention as an alternative to electron-driven and collision induced dissociation methods. Absorption of the high-energy UV photon is dispersed over the whole peptide or protein and stimulates extensive C?Ca backbone fragmentation while the low-energy IR photons gradually increases the internal energy and thus favorably dissociates the most labile amide (C?N) bonds. This thesis focuses on the method development and applications for characterizing biomolecules by photon-based activation methods. The interest of combining high-energy UV photons and low-energy IR photons in an Orbitrap mass spectrometer, for protein and post-translationally modified peptide characterization, has been evaluated. Moreover, infrared multiphoton dissociation has been implemented in a gated electrostatic ion trap to push forward the limit of fragmentation methods to large megadalton ions. One of the main breakthroughs in this thesis is the ability to adapt these method developments and applications to biomolecular objects ranging from small peptides (in kilodalton mass range) to entire protein fibrils (in megadalton mass range)