In this work, the Collision Induced Dissociation under Energy Control method was extended to study the fragmentation of gas-phase biomolecules adenine (H5C5N5) and porphyrin FeTPPCl (C44H28N4FeCl). The population distribution for each dissociation channel has been experimentally determined as a function of the excitation energy of the parent molecular ions at a well-determined initial charge state. In collisions between Cl+ and adenine (Ade) at 3keV, the fragmentation pattern of Ade2+ is dominated by the loss of H2CN+ and the successive emission of HCN. The energy distribution of the parent dications confirms the successive emission dynamics. A specific decay channel is observed, i.e., the emission of a charged H2CN+ followed by the emission of HC2N2. In Kr8+-FeTPPCl collisions at 80keV, parent ions FeTPPCl1+,2+,3+ are observed, along with the corresponding decay patterns. It is found that in the first step the dominant low-energy-cost decay channel is the emission of Cl0 independent of the initial charge state of FeTPPClr+ (r=1-3). For the resulted dication FeTPP2+, the dominant fragmentation channel is the neutral evaporation; for the trication however, the dominant fragmentation channel is the asymmetrical fission. In the case of H+ and F+ impact at 3keV, due to the different reaction windows opened in the two collision systems, different fragmentation patterns are observed. Furthermore, nH2 loss processes are observed. Additionally, the production yield of the negative ion emerged in F2+-Ade collision at 30keV is measured to be about 1% using a new experimental approach.

Cytochrome P450 (CYP) family 4 constitutes monoxygenases responsible for hydroxylation of fatty acids and other lipids. For example, CYP4F3 metabolizes leukotrienes and CYP4F8 prostaglandin H. Importantly, six of the twelve CYP4 enzymes are orphans, i.e., with an unknown biological function. The catalytic activity of the enzyme CYP4F8 is known in seminal vesicles, but not in skin or psoriatic lesions, where CYP4F8 is highly expressed. The orphan CYP4F22 is also expressed in skin, and mutations in its gene has been linked to the rare skin disease lamellar ichthyosis, together with, inter alia, mutations in the genes of 12R-LOX and eLOX3. These enzymes appear to constitute a pathway producing hydroperoxides and epoxyalcohols from arachidonic acid. CYP4F22 is hypothesized to act in a consecutive step within this pathway. The aim of this thesis was to develop analytical methods to prepare and analyze hydroperoxides and epoxyalcohols derived from fatty acids by LC-MS/MS, and to investigate the catalytic performance of CYP4F8 and CYP4F22 for these substrates. The 12R-hydroperoxide of arachidonic acid (12R-HPETE) was prepared by autoxidation and separated from other hydroperoxides by chiral HPLC. MS/MS analysis showed that the hydroperoxides were unstable within the ion trap, but were stabilized by an increase in the isolation width. From the hydroperoxides, epoxyalcohols were generated by hematin treatment, and separated by normal phase HPLC. MS/MS spectra of several epoxyalcohols, derived both from arachidonic acid and linoleic acid, were characterized with aid of [2H]isotopomers and MS3 analysis. Apart from metabolic studies the thesis also include detailed information on MS/MS analysis of several oxygenated fatty acids, with proposed fragmentation mechanisms. The open reading frame of CYP4F22 was expressed in a recombinant yeast system, and LC-MS/MS analysis revealed that CYP4F22 catalyzed ω3 hydroxylation of arachidonic acid, but not any of the tested epoxyalcohols. In contrast, CYP4F8 metabolizes an epoxyalcohol derived from 12R-HPETE, 11R,12R-epoxy-10-hydroxyeicosatrienoic acid, to the ω3 hydroxy metabolite. Conclusively, it was demonstrated that LC-MS/MS could be used for the analysis and separation of hydroperoxides and epoxyalcohols for metabolic studies.

Ce mémoire présente une étude expérimentale de la fragmentation en phase gazeuse des biomolécules, adénine (H5C5N5) et porphyrine FeTPPCl (C44H28N4FeCl), induite par collision avec des ions à basse énergie. La distribution de population pour chaque voie de dissociation a été mesurée en fonction de l'énergie d'excitation des ions moléculaires parents avec la méthode CIDEC (Collision Induced Dissociation under Energy Control). Dans les collisions entre Cl+ à 3keV et adénine (Ade), le schéma de fragmentation de Ade2+ est dominée par la perte de H2CN+ et les émissions successives de HCN. La distribution de l'énergie des Ade2+ parents confirme la dynamique des émissions successives. Une voie de dissociation spécifique, à savoir l'émission successive de H2CN+ et HC2N2 est observée. Les schémas de fragmentation des ions moléculaires FeTPPCl1+, 2+, 3+ sont étudiés dans des collisions avec Kr8+ à 80 keV. Il est constaté qu’indépendante de l'état de charge initiale de FeTPPClr+ (r=1, 2, 3), la perte de Cl0 constitue la première étape de la chaîne de dissociation, tandis que l’état de charge initiale des molécules joue un rôle important dans les étapes suivantes de la dissociation. Dans les collisions avec H+ et F+ à 3keV, dû à un effet de fenêtre de réaction dans les processus de production d’ions négatifs, des schémas de fragmentation très différents sont observés pour FeTPPCl2+. Grâce à la mesure de l’énergie interne des molécules parents, la perte de nH2 est observée et analysée. De plus, le rendement de production d'ions négatifs, mesuré à environ 1% dans des collisions F2+-Ade à 30 keV, est étudié dans ce travail en utilisant une nouvelle approche expérimentale. / In this work, the Collision Induced Dissociation under Energy Control method was extended to study the fragmentation of gas-phase biomolecules adenine (H5C5N5) and porphyrin FeTPPCl (C44H28N4FeCl). The population distribution for each dissociation channel has been experimentally determined as a function of the excitation energy of the parent molecular ions at a well-determined initial charge state. In collisions between Cl+ and adenine (Ade) at 3keV, the fragmentation pattern of Ade2+ is dominated by the loss of H2CN+ and the successive emission of HCN. The energy distribution of the parent dications confirms the successive emission dynamics. A specific decay channel is observed, i.e., the emission of a charged H2CN+ followed by the emission of HC2N2. In Kr8+-FeTPPCl collisions at 80keV, parent ions FeTPPCl1+,2+,3+ are observed, along with the corresponding decay patterns. It is found that in the first step the dominant low-energy-cost decay channel is the emission of Cl0 independent of the initial charge state of FeTPPClr+ (r=1-3). For the resulted dication FeTPP2+, the dominant fragmentation channel is the neutral evaporation; for the trication however, the dominant fragmentation channel is the asymmetrical fission. In the case of H+ and F+ impact at 3keV, due to the different reaction windows opened in the two collision systems, different fragmentation patterns are observed. Furthermore, nH2 loss processes are observed. Additionally, the production yield of the negative ion emerged in F2+-Ade collision at 30keV is measured to be about 1% using a new experimental approach.

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