5-oxoETE triggers nociception in constipation-predominant irritable bowel syndrome through MAS-related G protein-coupled receptor D.

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder that is characterized by chronic abdominal pain concurrent with altered bowel habit. Polyunsaturated fatty acid (PUFA) metabolites are increased in abundance in IBS and are implicated in the alteration of sensation to mechanical stimuli, which is defined as visceral hypersensitivity. We sought to quantify PUFA metabolites in patients with IBS and evaluate their role in pain. Quantification of PUFA metabolites by mass spectrometry in colonic biopsies showed an increased abundance of 5-oxoeicosatetraenoic acid (5-oxoETE) only in biopsies taken from patients with IBS with predominant constipation (IBS-C). Local administration of 5-oxoETE to mice induced somatic and visceral hypersensitivity to mechanical stimuli without causing tissue inflammation. We found that 5-oxoETE directly acted on both human and mouse sensory neurons as shown by lumbar splanchnic nerve recordings and Ca2+ imaging of dorsal root ganglion (DRG) neurons. We showed that 5-oxoETE selectively stimulated nonpeptidergic, isolectin B4 (IB4)-positive DRG neurons through a phospholipase C (PLC)- and pertussis toxin-dependent mechanism, suggesting that the effect was mediated by a G protein-coupled receptor (GPCR). The MAS-related GPCR D (Mrgprd) was found in mouse colonic DRG afferents and was identified as being implicated in the noxious effects of 5-oxoETE. Together, these data suggest that 5-oxoETE, a potential biomarker of IBS-C, induces somatic and visceral hyperalgesia without inflammation in an Mrgprd-dependent manner. Thus, 5-oxoETE may play a pivotal role in the abdominal pain associated with IBS-C.BBSRC BB/R006210/1 to James R F Hockley and Ewan St John Smith Rosetrees 834 Postdoctoral Grant (A1296) awarded to James R F Hockley and Ewan St John Smit

Study of protein modification induced by cigarette smoke

La fumée de cigarette, reconnue comme étant carcinogène, est un aérosol complexe composé de plusieurs milliers de substances chimiques susceptibles de réagir avec les biomolécules. Aussi l’identification des modifications post-traductionnelles des protéines induites par la fumée de cigarette s’avère être un enjeu majeur à la compréhension de certaines pathologies telles que les cancers ou les maladies cardiovasculaires.L’objectif de ce travail a été de développer une méthode d’analyse basée sur la spectrométrie de masse à haute résolution afin d’identifier et de localiser les modifications des protéines exposées à la phase gazeuse de la fumée de cigarette. Deux techniques ont été utilisées : l’analyse MALDI-TOF-TOF et l’analyse nanoESI-FT-ICR couplée à la nano chromatographie liquide. Dans le but d’optimiser la réaction avec la fumée de cigarette, des modèles de réactions ont été développés sur des peptides et protéines standards. Ainsi des réactions d’oxydation et de nitration ont été effectués à partir des radicaux libres HO° et NO2° tandis que des réactions de formation d’adduits ont été réalisées à partir d’un équivalent d’aldéhyde (formaldéhyde, acétaldehyde, malonaldéhyde, acroléine, crotonaldéhyde et glyoxal). Les mêmes standards protéiques ont ensuite été exposés à la fumée de cigarette à l’aide d’une machine à fumer. Par comparaison avec les modèles de réaction, des adduits provenant de l’acétaldéhyde, de l’acroléine et du crotonaldéhyde ont pu être identifiés et localisés sur des résidus lysines spécifiques. Ces résultats ont permis de mettre en évidence que les protéines étaient majoritairement modifiées par les aldéhydes contenus dans la fumée de cigarette.Cigarette smoke, well known to be carcinogen, is a high complex aerosol composed of further thousands of chemicals able to react with biomolecules. Also the identification of post-translational protein modifications induced by cigarette smoke is a major stake for the understanding of some pathology like cancer and cardiovascular disease. The aim of this work was to develop an analytical method based on high resolution mass spectrometry in order to identify and localize protein modifications induced by cigarette smoke. Two techniques were used: a MALDI-TOF-TOF analysis and a on line nano liquid chromatography nanoESI-FT-ICR analysis. In order to optimize reaction with cigarette smoke, model reactions were developed on standard peptides and protein. Thus oxidation and nitration were realized using HO° and NO2° free radicals while formation of adducts was performed using 1 eq of aldehydes (formaldehyde, acetaldehyde, malonaldehyde, acrolein, crotonaldehyde and glyoxal). Same proteins were then exposed to cigarette smoke using a smoking machine.By comparison with model reactions, adduct coming from acetaldehyde, acrolein and crotonaldehyde were identified and localized on specific lysine amino acids. These results allowed highlighting that proteins are principally modified by aldehydes contained in cigarette smoke

Étude des modifications des protéines induites par la fumée de cigarette

La fumée de cigarette, reconnue comme étant carcinogène, est un aérosol complexe composé de plusieurs milliers de substances chimiques susceptibles de réagir avec les biomolécules. Aussi l identification des modifications post-traductionnelles des protéines induites par la fumée de cigarette s avère être un enjeu majeur à la compréhension de certaines pathologies telles que les cancers ou les maladies cardiovasculaires.L objectif de ce travail a été de développer une méthode d analyse basée sur la spectrométrie de masse à haute résolution afin d identifier et de localiser les modifications des protéines exposées à la phase gazeuse de la fumée de cigarette. Deux techniques ont été utilisées : l analyse MALDI-TOF-TOF et l analyse nanoESI-FT-ICR couplée à la nano chromatographie liquide. Dans le but d optimiser la réaction avec la fumée de cigarette, des modèles de réactions ont été développés sur des peptides et protéines standards. Ainsi des réactions d oxydation et de nitration ont été effectués à partir des radicaux libres HO et NO2 tandis que des réactions de formation d adduits ont été réalisées à partir d un équivalent d aldéhyde (formaldéhyde, acétaldehyde, malonaldéhyde, acroléine, crotonaldéhyde et glyoxal). Les mêmes standards protéiques ont ensuite été exposés à la fumée de cigarette à l aide d une machine à fumer. Par comparaison avec les modèles de réaction, des adduits provenant de l acétaldéhyde, de l acroléine et du crotonaldéhyde ont pu être identifiés et localisés sur des résidus lysines spécifiques. Ces résultats ont permis de mettre en évidence que les protéines étaient majoritairement modifiées par les aldéhydes contenus dans la fumée de cigarette.Cigarette smoke, well known to be carcinogen, is a high complex aerosol composed of further thousands of chemicals able to react with biomolecules. Also the identification of post-translational protein modifications induced by cigarette smoke is a major stake for the understanding of some pathology like cancer and cardiovascular disease. The aim of this work was to develop an analytical method based on high resolution mass spectrometry in order to identify and localize protein modifications induced by cigarette smoke. Two techniques were used: a MALDI-TOF-TOF analysis and a on line nano liquid chromatography nanoESI-FT-ICR analysis. In order to optimize reaction with cigarette smoke, model reactions were developed on standard peptides and protein. Thus oxidation and nitration were realized using HO and NO2 free radicals while formation of adducts was performed using 1 eq of aldehydes (formaldehyde, acetaldehyde, malonaldehyde, acrolein, crotonaldehyde and glyoxal). Same proteins were then exposed to cigarette smoke using a smoking machine.By comparison with model reactions, adduct coming from acetaldehyde, acrolein and crotonaldehyde were identified and localized on specific lysine amino acids. These results allowed highlighting that proteins are principally modified by aldehydes contained in cigarette smoke.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

Development of a method for quantitative profiling of the carnitine family by LC-HRMS

International audienceL-carnitine plays an essential role in tissue energy metabolism by transporting long-chain fatty acids (FAs) across the inner membrane of the mitochondria. The mitochondrial membrane is impermeable to long-chain fatty acids (12 to 20C) and L-carnitine is the only transporter that allows them to enter the mitochondria. The FAs, activated into Acyl-CoA, are converted into acylcarnitine and thus cross the mitochondrial membrane, with the action of three enzymes. Once in the mitochondria, the FAs, once again in the form of Acyl-CoA, undergo β-oxidation, the major role of which is to supply ATP to organs with high energy requirements. This metabolic pathway is a key player in metabolism.This poster presents the implementation of an extraction and quantitative analysis method specific to the carnitine family: from L-carnitine to C18:1 acylcarnitine, using liquid chromatography coupled to an orbitrap-type mass spectrometer (Thermo Exploris 240). Initial extraction tests were carried out on tissues and plasma, as well as a separation on a Waters Acquity HSS T3 column. Various deuterated internal standards (of varying chain lengths) were used for absolute quantification. The method will be validated.This new profiling will be offered as a service on MetaToul, but it will mainly be used to monitor these partially 13C-labelled compounds as part of metabolic labelling experiments. It will be an important complement to the profile of labelled FAs already on offer.La L-carnitine joue un rôle essentiel au niveau du métabolisme énergétique des tissus via le transport des acides gras (AG) à chaîne longue à travers la membrane interne de la mitochondrie. Cette dernière est imperméable aux AG à chaîne longue (12 à 20C) et la L-carnitine est l’unique transporteur leur permettant de pénétrer au sein de la mitochondrie. Les AG, activés en Acyl-CoA, sont convertis en acylcarnitine et traversent ainsi la membrane mitochondriale, avec l’action de trois enzymes. Une fois dans la mitochondrie, les AG, à nouveau sous forme d’Acyl-CoA, vont subir la β -oxydation, dont le rôle majeur est la fourniture d’ATP aux organes à forts besoins d’énergie. Cette voie métabolique est un acteur clé en métabolisme.Ce poster présente la mise en place d’une méthode d’extraction et d’analyse quantitative spécifiques de la famille des carnitines : de la L-carnitine jusqu’à l’acylcarntine C18:1, par chromatographie liquide couplée à un spectromètre de masse de type orbitrap (Thermo Exploris 240). Des premiers tests d’extraction ont été réalisés sur tissus et plasma ainsi qu’une séparation sur colonne Waters Acquity HSS T3. Différents étalons internes deutérés (de longueurs de chaines variés) ont été utilisés pour faire les quantifications absolues. Une validation de la méthode sera effectuée.Ce nouveau profilage sera proposé en prestations sur MetaToul mais il sera surtout décliné pour suivre ces composés partiellement marqués au 13C dans le cadre d’expérimentation de marquage métabolique. Il sera un complément important au profil d’AG marqués déjà proposé

Global analysis of lipids by SFC-HRMS (Q-Tof) : an analytical and data processing challenge

International audienc

Atypical cleavage of protonated N‐fatty acyl amino acids derived from aspartic acid evidenced by sequential MS3 experiments

International audienceLipidomics calls for information on detected lipids and conjugates whose structural elucidation by mass spectrometry requires to rationalization of their gas phase dissociations toward collision-induced dissociation (CID) processes. This study focused on activated dissociations of two lipoamino acid (LAA) systems composed of N-palmitoyl acyl coupled with aspartic and glutamic acid mono ethyl esters (as LAA(*D) and LAA(*E)). Although in MS/MS, their CID spectra show similar trends, e.g., release of water and ethanol, the [(LAA(*D/*E)+H)–C2H5OH]+ product ions dissociate via distinct pathways in sequential MS3 experiments. The formation of all the product ions is rationalized by charge-promoted cleavages often involving stepwise processes with ion isomerization into ion–dipole prior to dissociation. The latter explains the maleic anhydride or ketene neutral losses from N-palmitoyl acyl aspartate and glutamate anhydride fragment ions, respectively. Consequently, protonated palmitoyl acid amide is generated from LAA(*D), whereas LAA(*E) leads to the [*E+H–H2O]+ anhydride. The former releases ammonia to provide acylium, which gives the C n H(2n−1) and C n H(2n−3) carbenium series. This should offer structural information, e.g., to locate either unsaturation(s) or alkyl group branching present on the various fatty acyl moieties of lipo-aspartic acid in further studies based on MSn experiments

Heteroleptic Ruthenium(II) Complexes with Bathophenanthroline and Bathophenanthroline Disulfonate Disodium Salt as Fluorescent Dyes for In-Gel Protein Staining

The in-gel detection of proteins for various proteomic experiments is commonly done with the fluorescent Ru-II tris(bathophenanthroline disulfonate) complex (Ru(BPS)(3)), which is more cost-effective compared to commercial Ru-based formulations but requires tedious procedures for its preparation and strongly acidic staining conditions. Herein, we report the synthesis and characterization of heteroleptic Ru-II complexes Ru(BPS)(2)(BP) and Ru(BPS)(BP)(2) containing bathophenanthroline (BP) and bathophenanthroline disulfonate disodium salt (BPS) in comparison with Ru(BPS)(3). It was shown by fluorescent and UV-vis measurements that novel Ru-II complexes were excitable in both UV and visible light, close to emission bands of classical lasers, which is important for successful in-gel protein detection. Novel fluorescent dyes demonstrated improved protein detection in comparison with commercially available SYPRO Ruby staining solution. In addition, unlike commonly used staining protocols, staining with Ru(BPS)(BP)(2) can be performed at nearly neutral pH, thereby reducing artificial post-translational modifications (PTMs)

Effect of ischemia on intestinal CYP-derived eicosanoids production.

<p>Synthesis of eicosanoids from arachidonic acid (AA) was measured by liquid chromatography-tandem mass spectrometry in control mice (naïve and sham operated mice) and following 50 minutes of ischemia. Data represent means ± SEM of 6 to 8 mice per group. *p<0.05, and ***p<0.001 <i>versus</i> the corresponding sham operated group.</p