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Photon and electron action spectroscopy of trapped biomolecular ions - From isolated to nanosolvated species

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In an effort to understand the vast complexity of the underlying processes within a cell at a molecular level, the first step lies in revealing the fundamental physical and chemical properties, as well as the structure, of biopolymers (proteins and DNA). With the development of modern experimental techniques it has become possible to study these large molecules under well-defined conditions in the gas phase, by closely inspecting their interactions with energetic photons and electrons. In this Thesis, we present the experimental setups for the action spectroscopy of peptides, proteins and nucleotides, as well as the hydrated complexes (hydrated nucleotides), in the gas phase. We present the details and the operation of the two experimental setups based on coupling the linear quadrupole ion trap with: (1) a VUV or a soft X-ray synchrotron beamline and (2) a focusing electron gun. In the case (1), the existing experimental setup consisting of a commercial quadrupole ion trap mass spectrometer (LTQ XL from Thermo Scientific), equipped with an electrospray ion source, was coupled to the VUV beamline DESIRS and the soft X-ray beamline PLEIADES at the synchrotron SOLEIL (France). The setups were used to study the photo-induced ionization/fragmentation of trapped biopolymers and nanosolvated species. The results obtained with this setups include VUV action spectroscopy of protonated Leucine-Enkephalin peptide (both a monomer and a dimer) and a nanosolvated nucleotide Adenosine monophosphate (AMP), in (5-15) eV photon energy range. The inner-shell action spectroscopy in the soft X-ray energy range (around C and N K-edge), was performed for multiply charged precursor of Ubiquitin protein. The photo-dissociation and photo-fragmentation ion yields for several fragment ions from all above mentioned macromolecules were extracted and the obtained spectral features were discussed considering relevant photon-induced processes. In the case (2), new experimental setup was developed by coupling the same LTQ XL ion trap with a focusing electron gun, in order to perform an electron activation tandem mass spectrometry, as well as an electron-impact action spectroscopy of trapped biopolymer ions. The ion optic simulations using SIMION program were performed in order to investigate the propagation of the electron beam in the RF+DC ion trap. Tests measurements for electron-induced fragmentation of Substance P, Melittin and Ubiquitin are presented for the impact energy of 300 eV. Finally, we present the electron-impact inner-shell action spectroscopy of the multiply charged Ubiquitin protein, in the vicinity of C K-edge energies of (280-300) eV. The electron-impact results are compared with the soft X-ray photon-impact action spectroscopy results obtained for the same target.
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Thesis
Les tubulines sont des protéines impliquées dans des processus biologiques essentiels à la vie cellulaire. Elles sont polymodifiées en leurs extrémités C-terminales. Différentes techniques ont été utilisées pour caractériser les polymodifications des tubulines. Mais certaines difficultés persistent concernant l’indentification fine de plusieurs structures. Le couplage de spectrométrie de masse à la mobilité ionique représente une avancée technique plus pertinente pour la séparation d’isomères de structures. En effet, la mobilité ionique peut séparer des ions de même rapport m/z en fonction de leur conformation. Dans la première partie de cette thèse, une analyse par mobilité ionique et spectrométrie de masse en tandem a permis la séparation de deux peptides de synthèse mimant des peptides C-terminaux de tubuline α diglycylés. L’hormone de croissance (GH) est une hormone anabolique et un agent dopant pour les sportifs. La disponibilité de la hGH recombinante (rhGH) dans le marché noir a augmenté la fréquence du dopage à la GH. Les tests antidopage approuvés par l’agence mondiale d’antidopage sont confrontés à certaines limites. Dans la deuxième partie de ma thèse, des analyses comparatives de la hGH naturelle et la rhGH ont été réalisées par spectrométrie de masse couplée à la chromatographie liquide en phase inverse pour trouver une différence chimique entre la hGH naturelle et la rhGH. La hGH naturelle extraite des glandes pituitaires de cadavres est glycosylée alors que la rhGH n’est pas modifiée. De manière intéressante, cette glycosylation se trouve sur un peptide protéospécifique de la hGH. Ce travail ouvre une piste pour le développement d’une nouvelle méthodologie pour les tests anti-dopage à la GH.
Article
A new apparatus was designed, coupling an electron gun with a linear quadrupole ion trap mass spectrometer, to perform m/z (mass over charge) selected ion activation by electron impact for tandem mass spectrometry and action spectroscopy. We present in detail electron tracing simulations of a 300 eV electron beam inside the ion trap, design of the mechanical parts, electron optics and electronic circuits used in the experiment. We also report examples of electron impact activation tandem mass spectra for Ubiquitin protein, Substance P and Melittin peptides, at incident electron energies in the range from 280 eV to 300 eV. Graphical abstract
Article
We have performed inner-shell electron impact action spectroscopy of mass and charge selected macromolecular ions. For this purpose, we have coupled a focusing electron gun with a linear quadrupoleion trapmass spectrometer. This experiment represents a proof of principle that an energy-tunable electron beam can be used in combination with radio frequency traps as an activation method in tandem mass spectrometry (MS2) and allows performing action spectroscopy. Electron impact MS2 spectra of multiply protonated ubiquitin protein ion have been recorded at incident electron energies around the carbon 1 s excitation. Both MS2 and single ionizationenergy dependence spectra are compared with literature data obtained using the soft X-ray activation conditions.
Article
We have studied the Vacuum Ultraviolet (VUV)photodissociation of gas-phase protonated leucine-enkephalin peptide ion in the 5.7 to 14 eV photon energy range by coupling a linear quadrupole ion trap with a synchrotron radiation source. We report VUV activation tandem mass spectra at 6.7, 8.4, and 12.8 eV photon energies and photodissociation yields for a number of selected fragments. The obtained results provide insight into both near VUV radiation damage and electronic properties of a model peptide. We could distinguish several absorption bands and assign them to particular electronic transitions, according to previous theoretical studies. The photodissociation yields appear to be very different for the various observed fragmentation channels, depending on both the types of fragments and their position along the peptide backbone. The present results are discussed in light of recent gas-phase spectroscopic data on peptides.
Article
DOI:https://doi.org/10.1103/PhysRev.65.343
Article
In this article we report new results for action spectroscopy of protonated peptide Leucine enkephalin (YGGFL). By coupling a linear ion trap mass spectrometer with a vacuum ultraviolet (VUV) synchrotron radiation beamline, we investigate photofragmentation pattern of this peptide, through the analysis of tandem mass spectra recorded over a range of VUV photon energies, below and above the ionization energy. The obtained fragmentation patterns are discussed and compared to previous results.
Article
From a theory of Hohenberg and Kohn, approximation methods for treating an inhomogeneous system of interacting electrons are developed. These methods are exact for systems of slowly varying or high density. For the ground state, they lead to self-consistent equations analogous to the Hartree and Hartree-Fock equations, respectively. In these equations the exchange and correlation portions of the chemical potential of a uniform electron gas appear as additional effective potentials. (The exchange portion of our effective potential differs from that due to Slater by a factor of 23.) Electronic systems at finite temperatures and in magnetic fields are also treated by similar methods. An appendix deals with a further correction for systems with short-wavelength density oscillations.
Article
Understanding the correlation between proteins' tertiary and electronic structures is a great challenge, which could possibly lead to a more efficient prediction of protein functions in living organisms. Here, we report an experimental study of the interplay between electronic and tertiary protein structure, by probing resonant core excitation and ionization over a number of charge-state selected precursors of electrically charged proteins. The dependence of the core ionization energies on the protein charge state shows that the ionization of a protonated protein is strongly correlated to its tertiary structure, which influences its effective Coulomb field. On the other hand, the electronic core-to-valence shell transition energies are not markedly affected by the unfolding of the protein, from compact to totally elongated structures, suggesting that frontier protein orbitals remain strongly localized. Nevertheless, the unfolding of a protein seems to influence the cross section ratio between different