Hypertension Is Associated with Marked Alterations in Sphingolipid Biology: A Potential Role for Ceramide

Background Hypertension is, amongst others, characterized by endothelial dysfunction and vascular remodeling. As sphingolipids have been implicated in both the regulation of vascular contractility and growth, we investigated whether sphingolipid biology is altered in hypertension and whether this is reflected in altered vascular function. Methods and Findings In isolated carotid arteries from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats, shifting the ceramide/S1P ratio towards ceramide dominance by administration of a sphingosine kinase inhibitor (dimethylsphingosine) or exogenous application of sphingomyelinase, induced marked endothelium-dependent contractions in SHR vessels (DMS: 1.4±0.4 and SMase: 2.1±0.1 mN/mm; n = 10), that were virtually absent in WKY vessels (DMS: 0.0±0.0 and SMase: 0.6±0.1 mN/mm; n = 9, p Conclusions Hypertension is associated with marked alterations in vascular sphingolipid biology such as elevated ceramide levels and signaling, that contribute to increased vascular tone

Spectroscopie Infrarouge d'Intermédiaires Réactionnels Organométalliques

This work is about a new methodology to structurally characterize organometallic reactive intermediates, and more generally molecular ions in the gas phase. Modern ion sources in mass spectrometry enable the transfer of chemical species from solution to the gas phase, and an emerging spectroscopic technique, IRMPD (InfraRed Multiple Photon Dissociation), is used to characterize their structure.We developed this technique by coupling two mass spectrometers (an ICR ion trap and a Paul type quadrupole ion trap) to the free electron laser located at Orsay. This infrared source is powerful enough to induce a resonant multiple photon absorption, and its tunability in the infrared (700-2000cm-1) was exploited to characterize a large variety of mass selected ions, in particular organometallic species.Part of this work was dedicated to the optimisation of the two experimental set-ups, as well as to the simulation of IRMPD spectra. We show that those latter are very similar to the infrared absorption spectra calculated with the hybrid density functional B3LYP. We show that IRMPD spectroscopy enables the characterization of the metal spin state and coordination mode for a polydentate ligand in reactive organometallic species that are very difficult to characterize in the condensed phase.Amine allylation by allylic alcohols, catalysed by palladium complexes, has been investigated. Several catalytic cycles were proposed, and the IRMPD spectra of the observed reactive intermediates allow for their structural characterization and a catalytic cycle is validated.Cette thèse propose une nouvelle méthode pour caractériser la structure d'intermédiaires réactionnels organométalliques et, en général, d'ions moléculaires en phase gazeuse. Les techniques modernes d'ionisation en spectrométrie de masse permettent le transfert des espèces chimiques de la solution vers la phase gazeuse, et une technique spectroscopique émergente dite IRMPD (InfraRed Multiple Photon Dissociation) est utilisée pour caractériser leur structure.Nous avons développé cette technique avec deux spectromètres de masse (un piège ICR et un piège quadripolaire de Paul) couplés au laser à électrons libres d'Orsay. Cette source infrarouge a l'intensité requise pour induire l'absorption résonante de multiples photons, et son accordabilité dans l'infrarouge (700-2200cm-1) a été exploitée pour caractériser une grande variété d'ions sélectionnés en masse, en particulier des systèmes organométalliques.Une partie de cette thèse a été dédiée à la mise au point des deux montages expérimentaux, ainsi qu'à la modélisation des spectres IRMPD. Nous montrons que ceux-ci sont très semblables aux spectres infrarouges d'absorption calculés à l'aide de la fonctionnelle de la densité B3LYP. Nous montrons que l'IRMPD permet de caractériser le spin du métal et le mode de coordination d'un ligand polydentate dans des espèces organométalliques réactives très difficiles à caractériser en phase condensée.La réaction d'allylation des amines par un alcool allylique, catalysée par un complexe du palladium, a été étudiée. Plusieurs cycles catalytiques sont proposés, et le spectre IRMPD des intermédiaires réactionnels observés permet, en caractérisant leur structure, de valider un cycle catalytique

Combining Structural Probes in the Gas Phase - Ion Mobility-Resolved Action-FRET

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Structure of Pb²⁺-deprotonated dGMP complexes in the gas phase: a combined MS-MS/IRMPD spectroscopy/Ion Mobility study

The authors wish to thank the CLIO team (J. M. Ortega, C. Six, G. Perilhous, J. P. Berthet) as well as P. Maitre and V. Steinmetz for their support during the experiments.International audienceThe structure of the Pb2+–deprotonated 2′-deoxyguanosine-5′-monophosphate (dGMP) complex, generated in the gas phase by electrospray ionization, was examined by combining tandem mass spectrometry, mid-infrared multiple-photon dissociation (IRMPD) spectroscopy and ion mobility. In the gas phase, the main binding site of Pb2+ onto deprotonated dGMP is the deprotonated phosphate group, but the question is whether an additional stabilization of the metallic complex can occur via participation of the carbonyl group of guanine. Such macrochelates indeed correspond to the most stable structures according to theoretical calculations. A multiplexed experimental approach was used to characterize the gas-phase conformation of the metallic complex and hence determine the binding mode of Pb2+ with [dGMP]−. MS/MS analysis, observation of characteristic bands by IRMPD spectroscopy, and measurement of the ion mobility collision cross section suggest that gaseous [Pb(dGMP)-H]+ complexes adopt a macrochelate folded structure, which consequently differs strongly from the zwitterionic forms postulated in solution from potentiometric studies

Couplage laser/mobilité ionique - fragmentation résolue en conformation

communication par afficheNational audienceLa détermination de la structure de systèmes moléculaires complexes tels que les protéines ou les complexes protéiques représente un enjeu important aussi bien pour la compréhension du fonctionnement de ces édifices au niveau moléculaire que pour le développement de stratégies d'identification ou de diagnostic. Avec la taille croissante des systèmes d'intérêt, les techniques disponibles atteignent leur limite et la mise au point de méthodes alternatives devient nécessaire. Une démarche efficace consiste à combiner des techniques différentes donnant des informations complémentaires et orthogonales sur les molécules étudiées. C'est dans cet esprit que nous avons développé un nouveau dispositif expérimental qui permet de coupler un laser à un spectromètre par mobilité ionique. La spectrométrie par mobilité ionique consiste à séparer des ions selon leur vitesse de diffusion dans un gaz sous l'influence d'un champ électrique. A travers la mesure d'une section efficace de collision avec le gaz, cette technique constitue une sonde globale de la conformation en phase gazeuse des ions étudiés. Dans notre dispositif, le pouvoir de séparation de la mobilité ionique est utilisé pour sélectionner les molécules en conformation avant de provoquer leur fragmentation par absorption d'un ou plusieurs photons. L'objectif de ces expériences de fragmentation résolue en conformation est d'une part de comprendre l'influence de la conformation sur les canaux de fragmentation et d'autre part d'utiliser la sensibilité des sondes optiques à l'arrangement local des molécules pour remonter à une information structurale. La combinaison de sondes locale et globale de la conformation de biomolécules ouvre de nombreuses possibilités pour l'étude d'édifices moléculaires complexes en permettant avec un même instrument d'accéder à différents niveaux de structuration. Nous présenterons les premiers résultats obtenus sur des sucres et des peptides modifiés par greffage de chromophores. Ces systèmes modèles serviront de base à l'application de cette technique à des protéines entières et à des complexes protéine-ligand

Bringing Molecular Dynamics and Ion-Mobility Spectrometry Closer Together: Shape Correlations, Structure-Based Predictors, and Dissociation

International audienceUnfolding of proteins gives detailed information about their structure and energetics and can be probed as a response to a change of experimental conditions. Ion mobility coupled to native mass spectrometry is a gas-phase technique that can observe such unfolding in the gas phase by monitoring the collision cross section (CCS) after applying an activation, for example, by collisions (collision-induced unfolding, CIU). The structural assignments needed to interpret the experiments can profit from dedicated modeling strategies. While predictions of ion-mobility data for well-defined and structurally characterized systems is straightforward, systematic free-energy calculations or biased molecular dynamics simulations that employ IMS data are still limited. The methods with which CCS values are calculated so far do not allow for analytical gradients needed in biased molecular dynamics (MD), and further, explicit CCS calculations still can pose computational bottleneck—when integrated into MD-bioinformatics workflows. These limitations motivate one to revisit known correlations of the CCS with the aim to find computationally cheap and versatile but still at least semiquantitative descriptions of the CCS by pure structural descriptors. We have therefore investigated the correlation of CCS with the key structural parameter often used in computational unfolding studies—the gyration radius—for several small monomeric and dimeric proteins. We work out the challenges and caveats of the combinations of the configurational sampling method and the CCS-calculation algorithm. The correlations were found to be sensitive to the generation conditions and additionally to the system topology. To reduce the amount of fitting to be undertaken, we devise a simple structural model for the CCS that shares some commonalities with the hard-sphere model and the projection algorithm but is designed to take unfolding into account. With this model, we suggest a two-point interpolating function rather than fitting a large data set, at only little deterioration of the predictive power. We further proceed to a model with composition and structure dependence that builds only upon the gyration radius and the chemical formula to apply the found CCS scaling behavior—the scaled macroscopic sphere (sMS) predictor. We demonstrate its applicability to describe unfolding and also its transferability for a larger set of structures from the RSCPDB. As we have found for the dimeric systems, that shape correlations with one global descriptor qualitatively break down, we finally suggest a recipe to switch between global and fragment-based CCS prediction, that takes up the ideas of coarse-graining protein complexes. The presented models and approaches might provide a basis to boost the integration of structural modeling with multistage IMS experiments, especially in the field of large-scale bioinformatics or “on-the-fly” biasing of MD, where computational efficiency is critical

Ultraviolet, Infrared, and High-Low Energy Photodissociation of Post-Translationally Modified Peptides

International audienceMass spectrometry-based methods have made significant progress in characterizing post-translational modifications in peptides and proteins; however, certain aspects regarding fragmentation methods must still be improved. A good technique is expected to provide excellent sequence information, locate PTM sites, and retain the labile PTM groups. To address these issues, we investigate 10.6 μm IRMPD, 213 nm UVPD, and combined UV and IR photodissociation, known as HiLoPD (high-low photodissociation), for phospho-, sulfo-, and glyco-peptide cations. IRMPD shows excellent backbone fragmentation and produces equal numbers of N- and C-terminal ions. The results reveal that 213 nm UVPD and HiLoPD methods can provide diverse backbone fragmentation producing a/x, b/y, and c/z ions with excellent sequence coverage, locate PTM sites, and offer reasonable retention efficiency for phospho- and glyco-peptides. Excellent sequence coverage is achieved for sulfo-peptides and the position of the SO3 group can be pinpointed; however, widespread SO3 losses are detected irrespective of the methods used herein. Based on the overall performance achieved, we believe that 213 nm UVPD and HiLoPD can serve as alternative options to collision activation and electron transfer dissociations for phospho- and glyco-proteomics

Spectre infrarouge d'ions (nucléobase)H+ et [Pb(nucléobase)-H]+ produits par electrospray dans un piège ionique quadripolaire (ITMS)

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