Anionic metal-oxalate complexes have been generated in the gas phase and an attempt at determining plausible structures were made. Two different experimental techniques were coupled to mass spectrometry: Infrared Multiphoton Dissociation (IRMPD) and ion mobility. Both techniques were compared to theoretical structures calculated using various levels of theory. With the use of IRMPD, frequencies were generated for each complex and compared to theoretical frequencies. Plausible structures for all complexes were found using the M-series of density functional levels of the theory when the 6-311+gd basis set was used and Bhandhlyp functional was appropriate for the lanl2dz basis set. Using ion mobility allowed for collision cross-sections to be calculated and compared to theoretical collision cross-sections of the various structures. Unfortunately no plausible structures were determined using this technique due to a lack of calibrants for the negative mode of ion mobility.

Cette thèse présente une étude expérimentale et théorique de peptides protonés contenant plusieurs dizaines d’atomes en phase gazeuse. Le premier objectif de ce travail consistait à coupler trois techniques expérimentales complémentaires, la spectroscopie IRMPD, la spectrométrie de mobilité ionique et la dissociation induite par attachement d’électron (ECD), afin de caractériser la structure des peptides amyloïdes Aβ₁₂-₂₈ et de tryptophane zippers TZ1 et TZ4. L’originalité de cette étude porte sur l’analyse de la distribution d’intensité des fragments c/z obtenus en ECD en relation avec les informations structurelles obtenues par mobilité et IRMPD. Grâce à cette approche combinée, nous avons pu proposer les structures les plus probables adoptées en phase gazeuse par ces peptides flexibles. Nous avons montré que la structure native des peptides n’était pas conservée au passage en phase gazeuse. L’interprétation des données expérimentales repose sur un travail théorique important alliant des dynamiques moléculaires utilisant un champ de force AMBER aux méthodes de chimie quantique au niveau DFT pour simuler les spectres vibrationnels des peptides. Le second axe de ce travail a porté sur la conception, la réalisation et la caractérisation d’une nouvelle technique de mise en phase gazeuse de biomolécules et leurs complexes non-covalents par désorption laser IR sur une micro-gouttelette liquide directement sous vide. Cette technique originale pourrait être une alternative aux techniques ESI et MALDI. Nous avons obtenu les premiers spectres de masse et optimisé les paramètres importants de l’expérience : longueur d’onde, intensité laser, résolution en masse du spectromètre à temps de vol. D’autres développements sont en cours pour améliorer la détection et la résolution en masse de ce type de source qui produit des ions avec une grande dispersion en énergie cinétique : piéger les ions dans une trappe quadrupolaire et entraîner les ions dans une détente supersonique. / Gas-phase studies of large biological molecules have emerged with the advent of soft production methods of biomolecular ions under vacuum (like ESI or MALDI) combined to mass spectrometry. The first aim of this work was to use three complementary experimental techniques, namely IRMPD spectroscopy, ion mobility mass spectrometry and electron capture dissociation ECD to probe the gas-phase structures of amyloïd Aβ₁₂-₂₈ and tryptophan zippers TZ1 and TZ4 peptides. The main originality of this study is to analyse the specific c/z product ion abundances in ECD experiments with the structural information gained through IR spectroscopy and ion mobility experiments. With this complementary approach, we were able to assign the most probable gas-phase structures of these flexible peptides. In particular, it is shown that the native structure of the peptides is not conserved in the gas phase. This study relies on the interplay between experiments and theoretical calculations. To that end, we used several theoretical methods, ranging for molecular dynamics using a classical force field (AMBER) to quantum mechanics calculations to simulate the vibrational spectra of the peptides. The second aim of this work was to design, develop and optimize a unique device, coupling a novel source introducing biomolecules in the gas phase, based on laser desorption from liquid micro-droplets directly into vacuum, coupled with a time-of-flight mass spectrometer. This desorption source is an original alternative to the usual methods (ESI or MALDI). Through mass spectrometry techniques, it should allow investigating the complexation processes of non-covalently bound species in conditions as close as the ones encountered in solution. We have recently obtained the first mass spectra. Due to the high velocity spread of the desorbed ions, new developments are needed to improve the mass resolution. In particular, we plan to transfer the ions in a quadrupole ion trap and to perform the laser desorption in front a pulsed valve to stream the ions in the supersonic expansion.

Este trabalho conta com um apanhado geral da técnica de espectrometria de massas acoplada a espectroscopia vibracional de íons, suas aplicações, limitações, desenvolvimento e embasamento teórico. Para tal é apresentado um resumo da teoria quântica aplicada à espectroscopia vibracional, natureza da luz, eletromagnetismo e ótica de sistemas de OPO/OPA, laser de gás e de estado sólido. A seção experimental é dividida em duas. A primeira apresenta o acoplamento físico do espectrômetro de massas às fontes de radiação laser, juntamente com os softwares para permitir comunicação entre esses módulos. Na segunda parte, são apresentados espectros IRMPD de amostras padrão para comparação e validação com a literatura bem como espectros inéditos para exemplificar o uso da técnica de espectroscopia de íons em sistemas de interesse biológico, como as bases nitrogenadas do DNA. / This master thesis presents a general overview of the mass spectrometry technique coupled to vibrational ion spectroscopy, its applications, limitations, development and theoretical basis. For that, a summary of the quantum theory applied to vibrational spectroscopy, the nature of light, electromagnetism and optics of OPO/OPA, gas discharge and solid-state laser systems is shown. The experimental section is divided in two, the first presents the physical coupling of the mass spectrometer to the laser sources, along with softwares which allows the communication between these modules. In the second part, some IRMPD spectra of standard samples are presented for comparison and validation with the literature as well as unpublished spectra to exemplify the use of the ion spectroscopy technique in systems of biological interest, such as the DNA bases.

Selective gas-phase dissociation strategies have been developed for the characterization of cross-linked peptides and proteins in quadrupole ion trap mass spectrometers. An infrared chromogenic cross-linker (IRCX) containing a phosphotriester afforded rapid differentiation of cross-linked peptides from unmodified ones in proteolytic digests of cross-linked proteins by selective infrared multiphoton dissociation (IRMPD). Only the cross-linked peptides containing the chromogenic phosphate underwent IRMPD and unmodified peptides were not affected by IR irradiation. IRMPD of IRCX-cross-linked peptides yielded uncross-linked y-ion sequence tags of the constituent peptides due to secondary dissociation of all primary product ions which contained the chromophore, thus allowing successful de novo sequencing of the cross-linked peptides. Peptides cross-linked via a two-step conjugation strategy through the formation of a bis-arylhydrazone (BAH) bond were selectively dissociated by ultraviolet radiation at 355 nm. The BAH-cross-linked peptides could be distinguished from not only unmodified peptides but also dead-end modified peptides based on the selectivity of ultraviolet photodissociation. In a complementary approach, electron transfer dissociation of BAH-cross-linked peptides resulted in preferential cleavage of the hydrazone bond which produced two modified peptides. These modified peptides were subsequently interrogated by CID which allowed for the original site of cross-linking to be pinpointed. IRMPD was implemented in a dual pressure linear ion trap to demonstrate successful photodissociation of peptides having modest absorptivities. Peptides were observed to efficiently dissociation by IR irradiation exclusively in the low pressure cell whereas no dissociation was observed in the high pressure cell due to extensive collisional cooling. IRMPD provided greater sequence coverage of the peptides than CID and yielded product ion mass spectra which were predominantly composed of singly charged product ions which simplified spectral interpretation. IRMPD was further applied for the sequencing of small-interfering RNA. Complete sequence coverage was obtained and the results were compared to CID. / text

Plusieurs facteurs conduisent à la génération de radicaux libres oxydants dans les organismes qui endommagent les biomolécules et en particulier les protéines du vivant. L’une des cibles de l’oxydation dans les protéines est la méthionine (Met). Son oxydation provoque des effets très dommageables, comme la maladie d’Alzheimer ou les maladies à prion. Nous avons étudié la réaction des radicaux hydroxyle (•OH) et de l’état triplet de la 3-carboxybenzophénone (3CB*) avec ces peptides. Le but de ce travail était de caractériser les espèces transitoires et les produits stables formés après oxydation radiolytique et photolytique de peptides contenant la Met (les dipeptides contenant de la Met et les pepetides plus longs tels que la bradykinine, une hormone humaine importante impliquée dans la diminution de la pression artérielle). Nous avons utilisé la photolyse éclair laser (LFP) et la radiolyse pulsée (PR) (pour les espèces transitoires de courte durée de vie), tandis que la radiolyse gamma et la photolyse continue (pour obtenir une caractérisation des produits stables). Les modifications structurelles ont été caractérisées par des techniques de HPLC et par couplage de la spectrométrie de masse (MS) et la détection par spectrométrie infrarouge couplée à la MS (IRMPD, CLIO laser à électrons libres). En outre, il nous a paru intéressant d’étudier deux autres dérivés de la benzophénone (BP), l’oxybenzone (OXB) et le sulisobenzone (SB), qui sont largement utilisés dans les produits de protection solaire commerciaux. En effet, l’application d’écrans solaires est controversée car certaines études épidémiologiques ont indiqué un risque accru de mélanome malin pour leurs utilisateurs. L’oxydation de dipeptides contenant Met par les radicaux •OH ou photosensibilisée par la ³(CB)* a conduit à la formation de radicaux cations centrés sur le soufre de la Met (>S•⁺) qui ont été en outre stabilisés par la formation de liaison deux centres à trois électrons (S∴Y)⁺, Y étant un atome possédant un doublet libre, ou qui ont subi une déprotonation donnant les radicaux contrés sur le carbone en α (α-S). L’oxydation des dipeptides par •OH a abouti à la formation de sulfoxyde de Met (MetSO) en tant que produit principal. Sans aucun doute, l’identification et la caractérisation des MetSO en solutions désoxygénées contenant la catalase est une étape importante dans la quête de produits stables. Toutefois, dans certains cas, d’autres produits ont été identifiés. En ce qui concerne, les produits stables de photolyse, ce sont des adduits avec le groupement 3CB, probablement résultant de la réaction de recombinaison radical-radical. Un autre produit formé au cours de la photolyse était 3CB-3CB résultant d’une photo-addition, qui a une structure similaire à celle du produit d’irradiations de la BP. Tous les produits identifiés (MetSO et la photo-adduits) ont été formés à partir des radicaux α-S par l’intermédiaire d’une dismutation ou une réaction avec 3CBH•/3CB•⁻. L’oxydation de la Met-Lys-bradykinine (MKBR) a abouti à la formation de photo-adduits similaires par réaction sensibilisée avec 3CB. L’oxydation induite de MKBR par •OH a abouti à plusieurs produits, en accord avec la non sélectivité des radicaux •OH. L’un des principaux produits est le MetSO et la phénylalanine hydroxylée. Notons que l’arginine n’est pas oxydée. Enfin, la photolyse de SB et OXB a été étudiée à l’aide de photolyse éclair au laser femto-et nanoseconde, ainsi que l’oxydation à un électron de ces molécules par radicaux •OH ont été réalisées en PR. Les résultats obtenus ont été comparés à ceux d’autres dérivés de la BP. L’état singulet excité subit un quenching à 100 % par transfert de proton intraomléculaire à l’état excité (ESIPT) en milieu aprotique et en milieu non polaire. Dans le cas d’un solvant polaire, la formation de radicaux phénoxyles a été identifiée. La réactivité des filtres solaires UV-excité vers dérivés simples de méthionine est également en cours d’étude. / Several factors (radiation, metabolism, pollutants) lead to the generation of oxidizing free radicals in living organisms that damage all biomolecules and especially proteins. One of the protein targets is Methionine (Met). Its oxidation causes highly damaging effects, such as Alzheimer’s or prion disease. The aim of this work was to investigate the transient species and the stable products formed after radiolytic and photolytic oxidation of Met-containing peptides. The reaction of hydroxyl radicals (•OH) and 3-carboxybenzophenone triplet state with Met-residue in peptides was investigated for model compounds (Met-dipeptides) and for longer peptides (e.g. Bradykinin). Laser flash photolysis and pulse radiolysis were used to characterize short-lived transient species, while gamma radiolysis and steady-state photolysis were used for quantitative and qualitative characterization of stable products. The structural modifications induced by oxidation have been characterized by the HPLC coupled with mass spectrometry and Infrared Multi Photon Dissociation Spectroscopy (IRMPD, CLIO Free electron laser). The oxidation of investigated Met-containing compounds by •OH or 3CB* led to the formation of S-centered radical cation >S•+ on Met-residue, that were further stabilized by formation of two-centered three-electron bond (S∴Y)+ or underwent the deprotonation reaction yielding the α-(alkylthio)alkyl radicals (α-S). The oxidation of Met-containing dipeptides by •OH radicals yielded the formation of Met sulfoxide (MetSO) as a main product. Undoubtedly, the identification and characterization of MetSO in deoxygenated solutions containing catalase was a milestone in investigation of stable products. However, in some cases, other products were identified. The stable products of photolysis were adducts with 3-carboxybenzophenone moiety, resulting from radical recombination reaction. Another identified product formed during photolysis was 3CB-3CB benzpinacol photoadduct, which has similar structure to the product of BP irradiations. Identified products (MetSO and the photo-adduct) were formed from the α-S via disproportionation or reaction with 3CBH•/3CBH•⁻. The oxidation of Met-Lys-Bradykinin (MKBR) yielded formation of similar photo-adducts via sensitized reaction with the 3CB*. The •OH induced oxidation of MKBR yielded several products, e.g. the sulfoxide and hydroxylated phenylalanine. In addition, other derivatives of benzophenone (oxybenzone (OXB) and sulisobenzone (SB)) were investigated due. They are widely used in commercial sun-protecting products dp to their unique photophysical properties. However the application of sunscreens awakes controversies because some epidemiological studies indicated an increased risk of malignant melanoma for their users. Photo-instability of sunscreen filters would result in reduced protection and may produce reactive free radicals or mutagens. In addition, the reactions of the sunscreens with oxygen free radicals e.g. hydroxyl radicals are likely to arise and they were not yet sufficiently documented. Finally, the radiolytic and photolytic properties of SB and OXB were investigated using femto-and nanosecond laser flash photolysis. Pulse radiolysis studies of the oxidation of those molecules by •OH radicals were performed. The results obtained for SB and OXB were compared to several other benzophenone derivatives. The results shown the formation of excited singlet state that was deactivated efficiently via the Excited State Intramolecular Proton Transfer (ESIPT). In case of polar solvent, the formation of trace amounts phenoxyl radicals was identified, while for nonpolar media those radicals were not observed. The reactivity of UV-excited sun filters towards simple derivatives of Met was also investigated, however, this topic requires further and more detailed investigations.

Many anticancer therapies are based on the interaction of small molecule drugs with nucleic acids, particularly DNA. Electrospray ionization tandem mass spectrometry has established itself as an irreplaceable tool for the characterization of DNA adducts produced by alkylating agents, carcinogens, and antitumor drugs, in addition to the characterization of nucleic acid post-transcriptional modifications. ESI-MS was used to assess the non-covalent binding of a novel series of intercalating anthrapyrazoles to duplexes containing different sequences. Relative binding affinities paralleled the shift in melting point of the DNA duplexes measured from a previous study. Upon collisionally induced dissociation of the duplex/anthrapyrazole complexes, different binding strengths were discerned based on the fragmentation patterns. In addition, the interactions of a new series of sulfur-containing acridine ligands, some that functioned as alklyating mustards, with duplex DNA were also evaluated. Non-covalent and covalent binding of each ligand was determined, and the site of adduction (G > A) was revealed for the covalent modifications. The distribution of cross-linked products and mono-adducts by psoralen analogs was also monitored by both LC-UV and IRMPD-MS methods. Reactions at 5’-TA sites were favored over 5’-AT sites. The sites of interstrand cross-linking were determined by fragmentation of the duplex/psoralen complexes by infrared multiphoton dissociation (IRMPD). Ultraviolet photodissociation (UVPD) at 193 nm caused efficient charge reduction of deprotonated oligodeoxynucleotides via electron detachment. Subsequent CID of the charge-reduced oligodeoxynucleotides formed upon electron detachment, in a net process called electron photodetachment dissociation (EPD), resulted in a diverse array of abundant sequence ions which allowed the modification site(s) of three modified oligodeoxynucleotides to be pinpointed to a more specific location than by conventional CID. Electron transfer dissociation (ETD) caused efficient charge reduction of multi-protonated oligonucleotides. Subsequent CAD of the charge-reduced oligonucleotides formed upon electron transfer, in a net process termed electron transfer collision activated dissociation (ETcaD), resulted in rich backbone fragmentation, with a marked decrease in the abundance of base loss ions and internal fragments. ETcaD of an oligonucleotide duplex resulted in specific backbone cleavages, with conservation of weaker non-covalent bonds. In addition, IRMPD and UVPD were used to activate charge-reduced oligonucleotides formed upon electron transfer. ET-IRMPD afforded tunable characterization of the modified DNA and RNA, allowing for modified bases to be directly analyzed. ET-UVPD promoted higher energy backbone fragmentation pathways and created the most diverse MS/MS spectra. The numerous products generated by the hybrid MS/MS techniques (ETcaD, ET-IRMPD, and ET-UVPD) resulted in specific and extensive backbone cleavages which allowed for the modification sites of multiple oligonucleotides to be pinpointed. / text

Advances in science and technology during the past decade have greatly enhanced the level of the structural investigation of macromolecules – peptides and proteins. Biological mass spectrometry has become one of the most precise and sensitive techniques in peptide and protein analysis. However, increasing demands of biotechnological applications require further progress to be made. In the present thesis the development and improvement of peptide and protein characterization methods and techniques based on ion-electron and ion-photon reactions in electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry are described. The focus is on the development of the electron capture dissociation method, recently discovered by the group of professor McLafferty, into a high rate, efficient tandem mass spectrometrical technique. The rate and reliability of the electron capture dissociation technique were greatly increased by implementation of low-energy pencil electron beam injection systems based on indirectly heated dispenser cathodes. Further implementation of a hollow electron beam injection system combined, in a single experimental configuration, two rapid fragmentation techniques, high rate electron capture dissociation and infrared multiphoton dissociation. Simultaneous and consecutive irradiations of trapped ions with electrons and photons extended the possibilities for ion activation/dissociation reaction schemes and lead to improved peptide and protein characterization. Using these improvements, high rate electron capture dissociation was employed in time-limited experiments, such as liquid chromatography–tandem mass spectrometry and capillary electrophoresis-tandem mass spectrometry. The analytical applications of the developed techniques have been demonstrated in top-down sequencing of peptides and proteins up to 29 kDa, improved sequencing of peptides with multiple disulfide bridges and secondary fragmentation (w-ion formation), as well as extended characterization of peptide mixtures separated by liquid chromatography and capillary electrophoresis. For instance, the dissociation of peptides resulting from enzymatic digestion of proteins provided complementary structural information on peptides and proteins, as well as their post-translational modifications.