Dissociation channel dependence on peptide size observed in electron capture dissociation of tryptic peptides

International audienceElectron capture dissociation (ECD) of a series of five residue peptides led to the observation that these small peptides did not lead to the formation of the usual c/z ECD fragments, but to a, b, y and w fragments. In order to determine how general this behavior is for small sized peptides, the effect of peptide size on ECD fragments using a complete set of ECD spectra from the SwedECD spectra database was examined. Analysis of the database shows that b and w fragments are favored for small peptide sizes and that average fragment size shows a linear relationship to parent peptide size for most fragment types. From these data, it appears that most of the w fragments are not secondary fragments of the major z ions, in sharp contrast with the proposed mechanism leading to these ions. These data also show that c fragment distributions depend strongly on the nature of C-terminal residue basic site: arginine leads to loss of short neutral fragments, whereas lysine leads to loss of longer neutral fragments. It also appears that b ions might be produced by two different mechanisms depending on the parent peptide size. A model for the fragmentation pathways in competition is proposed. These relationships between average fragment size and parent peptide size could be further exploited also for CID fragment spectra and could be included in fragmentation prediction algorithms

Structures de complexes d'initiation de la traduction étudiée par échanges isotopiques couplés à la spectrométrie de masse FT-ICR

Le complexe d'initiation de la traduction 3 des eucaryotes (eIF3) joue un role central dans le réseau d'interaction des divers facteurs d'initiation de la traduction qui s'assemblent sur les ribosomes 40S, et participent aux différentes réactions au cours de la voie d'initiation de la traduction. Chez Saccharomyces cerevisiaea, ce complexe est composé de cinq sous-unités, qui sont toutes homologues avec les sous-unités de cœur du complexe eIF3 des mammifères, composé de 13 sous-unités. Un objectif majeur actuellement est d'obtenir une structure tridimensionnelle de ce complexe. Dans une première étape vers la résolution de cette structure, les efforts portent sur la détermination des régions de liaisons entre sous-unités, dont assez peu sont actuellement connues. La région d'interaction entre la sous-unité 3i et le domaine C-terminal extrémal de 3b a récemment été résolu par RMN et structure cristallographique. D'un autre côté, la région d'interaction entre 3i et 3g, bien que localisée à l'extrémité N-terminale de 3g, doit encore être précisée. Les échanges hydrogène/deutérium (HDX) se sont développés depuis les années 1990 comme outil d'analyse structurale de protéines et de complexes multiprotéiques. La spectrométrie de masse est couramment utilisée pour réaliser la mesure de l'échange. L'approche HDX la plus usuelle repose sur une mesure de masse de peptide marqués issus d'une digestion enzymatique des protéines d'intérêt afin de déterminer le contenu en deutérium ainsi que leur vitesse d'incorporation. Pour ce travail, un spectromètre de masse à ultra-haute résolution, de type FT-ICR 7T, a été utilisé conjointement avec une séparation nano-LC pour générer des données HDX-MS de haute qualité. La précision de mesure de masse d'un spectromètre de masse FT-ICR n'est pas suffisante en elle-même pour identifier de façon certaine les peptides issus d'une digestion à la pepsine, du fait de l'absence de spécificité de la pepsine. Nous avons en conséquence développé une approche statistique pour l'identification des peptides, en se basant sur la définition d'une valeur de probabilité d'occurrence pour un peptide donné dans une digestion à la pepsine. En combinaison avec la précision élevée sur la mesure de masse, ce seul critère permet une identification efficace des peptides, sans devoir recourir à une validation par MS/MS systématique. Cette méthode a été mise en application pour l'étude des régions de liaison dans les complexes eIF3i :b et eIF3i :g. Dans les deux cas, 3i a été surexprimée sous sa forme complète. Au contraire, pour 3b et 3g, seul un segment partiel de la protéine native, contenant le domaine présumé d'interaction a été surexprimé. La liste de référence des peptides présents permet une excellente couverture de séquence et une forte superposition entre séquences adjacentes, ce qui assure une élucidation de la structure avec une bonne résolution spatiale. Pour la liaison entre 3i et 3b, les régions d'interactions qui sont apparues dans des conditions en solution proches des conditions physiologiques sont cohérentes avec la structure proposée par une autre équipe au cours de la thèse, apportant des informations complémentaires à celles issues de la structure cristallographique dérivée de la phase solide. Pour la liaison entre 3i et 3g, la région d'interaction a été étudiée en l'absence de toute donnée structurale à l'échelle atomique pour 3g. Les résultats apportent une vision nouvelle sur la formation du complexe entre 3g et 3i, et pour la première fois les régions de liaisons exacts ont été mis en évidence.The eukaryotic initiation factor 3 (eIF3) complex plays a core role in the interaction network among several eIFs that assemble on the 40S ribosomes and participate in the different reactions throughout the translation initiation pathway. The Saccharomyces cerevisiaea eIF3 complex comprises five subunits, all of which are the core subunits of the mammalian eIF3 complex consisting of 13 subunits. Attempts to decipher its tridimensionnal structure are under way. A first path to study the structure of this complex is to complete the identification of binding regions, few of which are currently known. Recently, the interaction region between eIF3i and extreme C-terminal domain of eIF3b has been obtained through NMR and crystal structure. On the other hand, the interaction region between 3i and 3g, although located to the N-terminal domain of 3g still remains to be defined. Hydrogen/deuterium exchanges (HDX) have been developed for a long time and are widely used for structural studies of proteins and multiprotein complexes. It is commonly analyzed using mass spectrometry. The most classic standard HDX-MS approach consists in making a mass measurement of deuterium-labelled peptides from an enzymatic digestion of the protein of interest to determine the level and rate of deuterium incorporation. In this study, a high performance 7 T FT-ICR mass spectrometer was used in combination with nanoLC separation to acquire highly accurate HDX-MS data. The precision on the mass measurement of FT-ICR MS is by itself not sufficient to unambiguously identify peptides from a pepsin digest due to the lack of pepsin specificity. We therefore developed a statistical approach for peptide identification, based on a probability of occurrence value of a given peptide within a pepsin digest. In combination with high mass accuracy, this method allows efficient identification of the peptides, without additional need of MS/MS verification. This method has been applied on the study of the binding regions in the complexes of eIF3i:bC3 and eIF3i:gC1 C. Peptide reference lists with high sequence coverage and rich sequence superposition ensured structure elucidation with high spatial resolution. For the binding of 3i and 3b, the detailed interaction regions were unveiled for proteins in the solution phase which resembled the physiological condition and were coherent with the reported protein structure, thus provided complimentary information to the crystallographic structure in solid phase. For the binding of 3i and 3g, the interaction regions were studied with the absence of any atomic structural information of 3g. This provides significant insights of the complex formation of 3i and 3g, and for the first time the precise binding regions were successfully revealed.PALAISEAU-Polytechnique (914772301) / SudocSudocFranceF

Thermochemical properties of the ammonia-water ionized dimer probed by ion-molecule reactions.

International audienceThe thermochemical properties of some small clusters such as the (H2O)2*+ dimer have already been investigated by both experimental and theoretical methods. The recent method to selectively prepare the ammonia-water ionized dimer [NH3, H2O]*+ (and not its proton transfer isomer [NH4+, OH*]) allowed us to study its chemical reactivity. This study focuses on the charge and proton transfer pathways: Ion-molecule reactions in the cell of an FT-ICR mass spectrometer were carried out with a range of organic compounds. Examination of the reactivity of the [NH3, H2O]*+ ionized dimer versus ionization energy and proton affinity of the neutral reagents shows a threshold in the reactivity in both instances. This leads to a bracketing of thermochemical properties related to the dimer. From these experiments and in agreement with ab initio calculations, the adiabatic recombination energy of the [NH3, H2O]*+ dimer was evaluated at -9.38 +/- 0.04 eV. The proton affinity bracketing required the reevaluation of two reference gas-phase basicity values. The results, in good agreement with the calculation, lead to an evaluation of the proton affinity of the [NH2*, H2O] dimer at 204.4 +/- 0.9 kcal mol(-1). These two experimental values are respectively related to the ionization energy of NH3*+ and to the proton affinity of NH2* by the difference in single water molecule solvation energies of ionized ammonia, of neutral ammonia, and of the NH2* radical

Catalyzed isomerization and decarbonylation of ionized formic acid and dihydroxycarbene

International audienceCatalyzed conversion of HCOOH*+ into HOCOH* + was studied in the cell of a FT-ICR in the presence of different molecules. The reaction of HCOOH*+ with SO2, whose proton affinity (PA) lies between that of the HOCO* radical at the carbon and at the oxygen sites, yields the HOCOH*+ carbene isomer as proved by its characteristic reaction with cyclopropane. When the PA of the catalyst lies above the highest PA of both sites of the HOCO* radical, formation of HOCOH*+ cannot be observed since its final state lies above that corresponding to protonation of the catalyst. However, reactions of DCO2H*+ and of HCO2D*+, which protonate several catalysts in an identical ratio which is very near of 1/1 at the beginning of the reaction, indicates that both ions, DCO2H*+ and of HCO 2D*+, convert into ion DO-C-OH*+ within a complex prior to protonation. The reactions of HCOOH*+ and HOCOH*+ with water were also more particularly studied by using theoretical calculations. Both reactions lead to protonated water and to the ionized water dimer which has been shown to possess the [H 2OH+?OH*] structure. The first step of the process is the conversion of the [HO(O)C*? H+?OH2] complex into [HOCO*?H +?OH2]. This latter complex undergoes two main pathways: on the one hand, it leads to protonation of water; on the other hand, it isomerizes to the [O*COH?H+?OH2] intermediate which dissociates to form [H2 OH+?OH*] with CO loss. Formation of H3O+ and [H2OH+?OH*] being rapid, the [HOCO*?H+?OH2] complex does not dissociate to yield the ionized carbene product which was not detected. Since the catalyzed isomerization of the 1,2-H transfer, converting HCOOH* + into HOCOH*+, is only observed within the corresponding complexes, this is a typical case of hidden isomerization. Finally, the differences in the unimolecular fragmentations of ionized formic acid and of its water solvated ion were explained. © 2003 Elsevier B.V. All rights reserved

Monitoring conformational landscape of ovine prion protein monomer using ion mobility coupled to mass spectrometry

Prion protein is involved in deadly neurodegenerative diseases. Its pathogenicity is linked to its structural conversion (alpha-helix to beta-strand transition). However, recent studies suggest that prion protein can follow a plurality of conversion pathways, which hints towards different conformers that might coexist in solution. To gain insights on the plasticity of the ovine prion protein (PrP) monomer, wild type (A136, R154, Q171), mutants and deletions of ARQ were studied by traveling wave ion mobility experiments coupled to mass spectrometry. In order to perform the analysis of a large body of data sets, we designed and evaluated the performance of a processing pipeline based on Driftscope peak detection and a homemade script for automated peak assignment, annotation, and quantification on specific multiply charged protein data. Using this approach, we showed that in the gas phase, PrPs are represented by at least three conformer families differing in both charge state distribution and collisional cross-section, in agreement with the work of Hilton et al. (2010). We also showed that this plasticity is borne both by the N- and C-terminal domains. Effect of protein concentration, pH and temperature were also assessed, showing that (1) pH does not affect conformer distributions, (2) protein concentration modifies the conformational landscape of one mutant (I208M) only, and (3) heating leads to other unfolded species and to a modification of the conformer intensity ratios

Transient multimers modulate conformer abundances of prion protein monomer through conformational selection

International audienc

A deconvolution method for the separation of specific versus nonspecific interactions in noncovalent protein-ligand complexes analyzed by ESI-FT-ICR mass spectrometry.

A method to separate specific and nonspecific noncovalent interactions observed in ESI mass spectra between a protein and its ligands is presented. Assuming noncooperative binding, the specific ligand binding is modeled as a statistical distribution on identical binding sites. For the nonspecific fraction we assume a statistical distribution on a large number of "nonspecific" interacting sites. The model was successfully applied to the noncovalent interaction between the protein creatine kinase (CK) and its ligands adenosine diphosphate (ADP) and adenosine triphosphate (ATP) that both exhibit nonspecific binding in the mass spectrum. The two sequential dissociation constants obtained by applying our method are K(1,diss) = 11.8 +/- 1.5 microM and K(2,diss) = 48 +/- 6 microM for ADP. For ATP, the constants are K(1,diss) = 27 +/- 7 microM and K(2,diss) = 114 +/- 27 microM. All constants are in good correlation with reported literature values. The model should be valuable for systems with a large dissociation constant that require high ligand concentrations and thus have increased potential of forming nonspecific adducts

Hydrogen radical abstraction by small ionized molecules, distonic ions and ionized carbenes in the gas phase

International audienceIn the gas phase and within ion-neutral complexes, H. abstraction by the ion from the neutral moiety was studied by using FT-ICR experiments and molecular orbital calculations. Ionized methanol abstracts rapidly H. from methane and other alkanes while its a-distonic counterpart, .CH2OH2+, is completely unreactive. On the other hand, ß-distonic ions, such as .CH2CH2XH+ (X=OCH3, NH2), are also unreactive towards methane but can abstract H . from ketones and ethers. Finally, ionized carbenes, such as HO-C-NH2.+, react with methane by a slow H. abstraction. Ab initio molecular orbital calculations at the G3(MP2) level were performed in order to understand these behaviors. For ionized methanol and its a-distonic counterpart, the reacting structure that could lead to H . abstraction is the highly stabilized complex between protonated methanol and a methyl radical, which yields the final state (CH 3OH2++.CH3) by simple cleavage. In the case of methanol the encounter complex with methane leads easily by rotation of the methane molecule to this reacting structure. In contrast, in the case of the a-distonic ion, the almost linear structure of the encounter complex [.CH2OH2+?CH4] and the high energy required for its isomerization into the reacting structure prevent the reaction. Two factors are required to observe H. abstraction in ß-distonic ions: the interaction energy of the encounter complex and the distance between the hydrogen to be transferred and the carbon radical. Reaction of the HO-C-NH 2.+ carbene with methane lies between these two extreme cases. The encounter complex is poorly stabilized (-8.7kcalmol-1) and the transition state for H. abstraction is very close to the reactants energy. © 2003 Elsevier B.V. All rights reserved

A prototype for catalyzed amide bond cleavage: Production of the [NH3, H2O](center dot+) dimer from ionized formamide and its carbene isomer

International audienceThe reaction of ionized formamide H2NCHO.+ with water leads to an exclusive loss of CO from the complex. This contrasts with the unimolecular reaction of low-energy ionized formamide, which loses exclusively one hydrogen atom. The unimolecular loss of CO is not observed because it involves several H-transfers corresponding to high-energy barriers. Experimental and theoretical studies of the role of solvation by water on the fragmentation of ionized formamide leads to three different results: (1) In contrast with different systems previously studied, in which solvation plays only a role on one or two steps of a reaction, a molecule of water is efficient in the catalysis of the decarbonylation process because water catalyzes all the steps of the reaction of ionized formamide, including the final dissociation of the amide bond. (ii) The catalyzed isomerization of carbonylic radical cations into their carbene counterparts is shown to be an important step in the process. To study this step, a precise probe, characterizing the carbene structure by ion-molecule reaction, is for the first time described. (iii) Finally, decarbonylation of ionized formamide yields the [NH3, H2O](.+) ion, which has not been generated and experimentally studied previously, By this method, the [NH3, H2O](.+) ion is generated in abundance and with a low internal energy content, allowing one either to prepare, by ligand exchange, a series of other solvated radical cations or to generate covalent structures such as distonic ions. First results on related systems indicate that the conclusions obtained for ionized formamide are widespread