Electron Capture in Charge-Tagged Peptides. Evidence for the Role of Excited Electronic States

International audienceElectron capture dissociation (ECD) was studied with doubly charged dipeptide ions that were tagged with fixed-charge tris-(2,4,6-trimethoxyphenyl)phosphonium-methylenecarboxamido (TMPP-ac) groups. Dipeptides GK, KG, AK, KA, and GR were each selectively tagged with one TMPP-ac group at the N-terminal amino group while the other charge was introduced by protonation at the lysine or arginine side-chain groups to give (TMPP-ac-peptide + H)2+ ions by electrospray ionization. Doubly tagged peptide derivatives were also prepared from GK, KG, AK, and KA in which the fixed-charge TMPP-ac groups were attached to the N-terminal and lysine side-chain amino groups to give (TMPP-ac-peptide-ac-TMPP)2+ dications by electrospray. ECD of (TMPP-ac-peptide + H)2+ resulted in 72% to 84% conversion to singly charged dissociation products while no intact charge-reduced (TMPP-ac-dipeptide + H)+• ions were detected. The dissociations involved loss of H, formation of (TMPP + H)+, and N–C(alpha) bond cleavages giving TMPP-CH2CONH2+ (c0) and c1 fragments. In contrast, ECD of (TMPP-ac-peptide-ac-TMPP)2+ resulted in 31% to 40% conversion to dissociation products due to loss of neutral TMPP molecules and 2,4,6-trimethoxyphenyl radicals. No peptide backbone cleavages were observed for the doubly tagged peptide ions. Ab initio and density functional theory calculations for (Ph3P-ac-GK + H)2+ and (H3P-ac-GK + H)2+ analogs indicated that the doubly charged ions contained the lysine side-chain NH3+ group internally solvated by the COOH group. The distance between the charge-carrying phosphonium and ammonium atoms was calculated to be 13.1-13.2 Å in the most stable dication conformers. The intrinsic recombination energies of the TMPP+-ac and (GK + H)+ moieties, 2.7 and 3.15 eV, respectively, indicated that upon electron capture the ground electronic states of the (TMPP-ac-peptide + H)+• ions retained the charge in the TMPP group. Ground electronic state (TMPP-ac-GK + H)+• ions were calculated to spontaneously isomerize by lysine H-atom transfer to the COOH group to form dihydroxycarbinyl radical intermediates with the retention of the charged TMPP group. These can trigger cleavages of the adjacent N–C(alpha) bonds to give rise to the c1 fragment ions. However, the calculated transition-state energies for GK and GGK models suggested that the ground-state potential energy surface was not favorable for the formation of the abundant c0 fragment ions. This pointed to the involvement of excited electronic states according to the Utah-Washington mechanism of ECD

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

Three-dimensional surface displacement of the 2008 May 12 Sichuan earthquake (China) derived from Synthetic Aperture Radar: evidence for rupture on a blind thrust

International audienceThe Sichuan earthquake,Mw7.9, struck the Longmen Shan (LMS) range front,China, on 2008 May 12, affecting an area of moderate historical seismicity where little active shortening has been previously reported. Recent studies based on space geodesy have succeeded in retrieving the far field surface displacements caused by the earthquake, but the near field (±25 km from the faults) coseismic surface displacement is still poorly constrained. Thus, shallow fault geometry and shallow coseismic slip are still poorly resolved. Here, for the first time for this earthquake, we combine C and L-band Synthetic Aperture Radar offsets data from ascending and descending tracks to invert for the 3-D surface displacement in the near coseismic field of the Sichuan earthquake. Our data, coupled with a simple elastic dislocation model, provide new results strongly suggesting the presence of a blind thrust striking along the range front and being active at depth during the earthquake. The presence of a rupture on a blind thrust brings new evidence for an out-of-sequence thrusting event and new elements for interpreting the tectonic strain partitioning in the LMS, which has important implications both for seismic hazard assessment and long-term evolution of the mountain belt

O-Glycosylation of the N-terminal region of the serine-rich adhesin Srr1 of Streptococcus agalactiae explored by mass spectrometry.

International audienceSerine-rich (Srr) proteins exposed at the surface of Gram-positive bacteria are a family of adhesins that contribute to the virulence of pathogenic staphylococci and streptococci. Lectin-binding experiments have previously shown that Srr proteins are heavily glycosylated. We report here the first mass-spectrometry analysis of the glycosylation of Streptococcus agalactiae Srr1. After Srr1 enrichment and trypsin digestion, potential glycopeptides were identified in collision induced dissociation spectra using X! Tandem. The approach was then refined using higher energy collisional dissociation fragmentation which led to the simultaneous loss of sugar residues, production of diagnostic oxonium ions and backbone fragmentation for glycopeptides. This feature was exploited in a new open source software tool (SpectrumFinder) developed for this work. By combining these approaches, 27 glycopeptides corresponding to six different segments of the N-terminal region of Srr1 [93-639] were identified. Our data unambiguously indicate that the same protein residue can be modified with different glycan combinations including N-acetylhexosamine, hexose, and a novel modification that was identified as O-acetylated-N-acetylhexosamine. Lectin binding and monosaccharide composition analysis strongly suggested that HexNAc and Hex correspond to N-acetylglucosamine and glucose, respectively. The same protein segment can be modified with a variety of glycans generating a wide structural diversity of Srr1. Electron transfer dissociation was used to assign glycosylation sites leading to the unambiguous identification of six serines and one threonine residues. Analysis of purified Srr1 produced in mutant strains lacking accessory glycosyltransferase encoding genes demonstrates that O-GlcNAcylation is an initial step in Srr1 glycosylation that is likely required for subsequent decoration with Hex. In summary, our data obtained by a combination of fragmentation mass spectrometry techniques associated to a new software tool, demonstrate glycosylation heterogeneity of Srr1, characterize a new protein modification, and identify six glycosylation sites located in the N-terminal region of the protein

Three dimensional surface displacement of the Sichuan earthquake (Mw 7.9, China) from Synthetic Aperture Radar

International audienceThe Sichuan earthquake, Mw 7.9, struck the Longmen Shan range front, in the western Sichuan province, China, on 12 May 2008. It severely affected an area where little historical seismicity and little or no significant active shortening were reported before the earthquake (e.g. Gu et al., 1989; Chen et al., 1994; Gan et al., 2007). The Longmen Shan thrust system bounds the eastern margin of the Tibetan plateau and is considered as a transpressive zone since Triassic time that was reactivated during the India-Asia collision (e.g., Tapponnier and Molnar, 1977, Chen andWilson 1996; Arne et al., 1997, Godard et al., 2009). However, contrasting geological evidences of sparse thrusting and marked dextral strike-slip faulting during the Quaternary along with high topography (Burchfiel et al., 1995; Densmore et al., 2007) have led to models of dynamically driven and sustained topography (Royden et al., 1997) limiting the role of earthquakes in relief building and leaving the mechanism of long term strain distribution in this area as an open question. Here we combine C and L band Synthetic Aperture Radar (SAR) offsets data from ascending and descending paths to retrieve the three dimensional surface displacement distribution all along the earthquake ruptures of the Sichuan earthquake. For the first time on this earthquake we present near field 3D co-seismic surface displacement, which is an important datum for constraining modelled fault geometry at depth. Our results complement other Interferometric Synthetic Aperture Radar (InSAR) and field analyses in indicating that crustal shortening is one of the main drivers for topography building in the Longmen Shan (Liu-Zeng, 2009; Shen et al., 2009; Hubbard and Shaw, 2009). Moreover, our results put into evidence a small but significant amount of displacement in the range front that we interpret as due to slip at depth on a blind structure. We verify this hypothesis by inverting the data against a simple elastic dislocation model.We discuss this result and its implications for understanding strain partitioning during the Sichuan earthquake

A visit to the Louvre: The Laboratoire de Recherche des Musées de France

International audienceNo Abstract Availabl

Dissociation par capture d'électrons peptides en phase gazeuse (mécanisme et application en aanalyse protéomique)

PALAISEAU-Polytechnique (914772301) / SudocSudocFranceF

Caractérisation de toxines peptidiques par spectrométrie de masse à haute résolution

PALAISEAU-Polytechnique (914772301) / SudocSudocFranceF

Electrophilic and radical reactivity of ionized aminohydroxycarbene toward alkenes

International audienceIon molecule reactions between ionized aminohydroxycarbene, [NH2COH](.+),1, and several representative alkenes (ethene, propene, 1-butene, 2-butene) were studied by Fourier transform ion cyclotron resonance mass spectrometry. Condensation processes were evidenced by the fragmentation pattern of the elusive collision complex which exhibits the characteristic behavior of the corresponding ionized aliphatic amide. Mechanistic pathways, corroborated by collision induced dissociation and deuterium labeling experiments and by G2 molecular orbital calculations, are proposed to explain the observed pattern. Proton and hydrogen atom transfer were also detected in several cases

Développement de nouvelles méthodologies par spectrométrie de masse à très haute résolution pour l analyse structurale de complexes protéiques

L'association échanges hydrogène/deutérium et spectrométrie de masse (HDX/MS) est devenue un outil analytique de choix pour l'étude de la structure et dynamique des protéines. L'approche classique "bottom-up" repose sur la digestion à la pepsine de la protéine deutérée et analyse des peptides protéolytiques par MS. Malheureusement, cette approche souffre de deux limitations majeures : (1) la résolution limitée par la taille des peptides après digestion et (2) l'échange inverse en solution avant l'analyse par MS. La MS en tandem classique ne peut pas être utilisée pour améliorer la résolution depuis que des expériences CID (Collision Induced Dissociation) sur des peptides deutérés ont montré une migration intramoléculaire des deutériums avant dissociation ("scrambling"). Par conséquent, il y a un grand intérêt à développer des approches HDX/MS alternatives pour améliorer les problèmes d'échange inverse, scrambling et résolution. Ce travail a donc consisté à mettre au point une méthode top-down HDX/MS robuste. Un nouveau système d'introduction des protéines deutérées dans le spectromètre de masse, appelé "cryosource", a été développé et les techniques d'activation basées sur la capture/transfert d'électron ont été utilisées. Il a récemment été montré que les méthodes ECD/ETD réduisaient le scrambling dans des conditions contrôlées avec une résolution à l'acide aminé près. L'utilisation de la cryosource a permis de diminuer l'échange inverse pendant des temps longs et avec des débits faibles. L'optimisation de tous les paramètres a été réalisée sur des peptides/protéine modèles. Notre objectif final était que notre approche soit applicable à l'analyse structurale du complexe aIF2The association of hydrogen/deuterium exchange and mass spectrometry (HDX/MS) has been emerged as a powerful analytical tool for probing structural and dynamic features of proteins. In classical bottom-up approach, the deuterated protein is digested by pepsin and the proteolytic digest is analyzed by mass spectrometry. Unfortunately, this approach suffers from two main drawbacks: (1) the resolution, which is limited by the size of the peptides after digestion and (2) the back-exchange that can take place in solution before the analysis by mass spectrometry. Classical tandem mass spectrometry cannot be used to improve resolution since Collision Induced Dissociation (CID) experiments on deuterated peptides have been shown for intramolecular deuterium migration before backbone cleavage ("scrambling"). Therefore, there is a great interest in the development of alternative HDX/MS approaches in order to improve back-exchange, scrambling and resolution problems. In this work, a complete optimized workflow for robust top-down HDX/MS has been set up. New system has been developed for introduction of the deuterated samples in the mass spectrometer, named "cryosource" and electron-based activation techniques, Electron Capture/Transfer Dissociation, were used. ECD and ETD have been shown recently to fragment, in proper experimental conditions, entire deuterated proteins with minimized scrambling and single residue resolution. The cryosource leads to negligible back-exchange during long periods of time and allows low flow rates. Optimization of all parameters was done using model peptides and protein. Our final goal was to use this methodology for the structural analysis of the complex aIF2.PALAISEAU-Polytechnique (914772301) / SudocSudocFranceF