Revisited BIA-MS combination: Entire "on-a-chip" processing leading to the proteins identification at low femtomole to sub-femtomole levels

International audienceWe present the results of a study in which biomolecular interaction analysis (BIA, Biacore 2000) was combined with mass spectrometry (MS) using entire "on-a-chip" procedure. Most BIA-MS studies included an elution step of the analyte prior MS analysis. Here, we report a low-cost approach combining Biacore analysis with homemade chips and MS in situ identification onto the chips without elution step. First experiments have been made with rat serum albumin to determine the sensitivity and validation of the concept has been obtained with an antibody/antigen couple. Our "on-a-chip" procedure allowed complete analysis by MS-MS of the biochip leading to protein identifications at low femtomole to sub-femtomole levels. Using this technique, identification of protein complexes were routinely obtained giving the opportunity to the "on-a-chip" processing to complete the BIA-MS approach in the discovery and analysis of protein complexe

GC-APPI-HRMS developments for the analysis of volatile organic compounds

International audienc

Des formules infantiles présentant des profils sensoriels différents

Cconférence de fin du projet Nativif financé par l'Institut Carnot Qualiment.National audienc

Flavour and Mass Spectrometry

International audienceThe flavour of food, commonly called ‘taste’, is an essential determinant of food choice by the consumer. Flavour perception in the mouth results from at least two sensory modalities, aroma and taste, due to odour-active and taste-active compounds released from the food matrix on eating. Instrumental assessment of flavour should characterise odouriferous molecules present in the volatile organic compounds of food and sapid components, essentially present in the non-volatile fraction of food. Mass spectrometry (MS) plays a fundamental role in the studies dedicated to flavour characterisation. Coupled with gas chromatography (GC) and benefitting from the latest development in multidimensional GC, MS is the method of choice to identify and quantify aroma compounds. Analytical advances for aroma compounds identification are presented, which include ionisation methods – electron ionisation and chemical ionisation – and mass analysers – tandem mass spectrometry (MS/MS) and high-resolution mass spectrometry – issues, not to forget hyphenation to olfactometry (GC-MS/O). Significant developments in the coupling of advanced MS instruments to liquid chromatography (LC-MS and LC-MS/MS) resulted in the possibility to take charge of non-volatile taste compounds. The great diversity of chemical structures responsible for taste, particularly bitterness, is emphasised in a comprehensive, while not exhaustive, review. Recent developments in direct-injection MS technologies, such as atmospheric pressure chemical ionisation and proton transfer reaction, have allowed developments in two kinds of applications. First, in vivo studies that allow taking charge online and real-time of the dynamic nature of flavour release in the mouth during consumption has received substantial attention. Second, untargeted fingerprinting of the volatiles emitted by foodstuffs (‘volatilome’), completed by more and more accessible non-volatiles profiling, has evolved in a metabolomics approach of flavour that could be called ‘flavouromics’. Both domains are reviewed, with examples taken from the most recent literature

Characterization of the aromatic potential of grape berry

National audienceIntroductionProbe is a research infrastructure gathering 4 analytical platforms of INRAE and allowing multi-approach and multi-complementarity to be explored and exploited. As a proof of concept, a common study was conducted around the structure and the evolution of the skin of the grape berry and the diffusion of compounds of interest during the winemaking process.Materials and methodsIn this context, the aromatic potential on two Vitis vinifera grape varieties (Carignan and Grenache,) were studied. Berries were harvested at an average potential alcohol of 12% vol. in the vineyard of the Pech Rouge experimental unit (INRAE, Gruissan, France) and separated according to their natural heterogeneity in terms of density (degree of maturity: D), yielding two batches (D−, D+). Stir Bar Sorptive Extraction (SBSE) was used to extract the volatile compounds from the free fraction and the bound volatile fraction after acid hydrolysis. The capillary GC analysis was carried out on a DB-Wax fused silica column and the mass spectrometer detector was operated in the full scan mode (m/z 29 to 350 uma) with electron ionization at 70 eV. Identification of unknown compounds was achieved by comparing the retention times of standards, and mass spectra in the Wiley/NIST/Inramass database.ResultsDifferentiation between berries and wines was the first source of variability in the samples and was found mainly due to the presence of alcohols, esters and acids in the wines (fermentative aromas produced by yeast) while the berries were rich in aldehydes. Regarding the effect of variety, Grenache appeared to be more aromatic than Carignan (used in blended wine), even if it is considered as a neutral grape. A significant effect of hydrolysis was mainly observed for vitispirane and linalool. As they were present in grapes as glycosidic precursors, their hydrolysis led to the formation of fragrant compounds that contribute to the aroma of wine. Finally, octanol, octanoic acid and hex-2-enal were more intense in D- berries, known to be characteristic of immature berries.ConclusionThe characterization of aromatic potential of grape berry was successfully investigated, showing differences between berries and wines, varieties and hydrolyse effect. Further work will consist in relating these results to the ones obtained by the other PROBE plateforms

Characterization and purification of a bacterial chlorogenic acid esterase detected during the extraction of chlorogenic acid from arbuscular mycorrhizal tomato roots

International audienceA Gram-negative bacterium able to grow using chlorogenic acid (5-caffeoylquinic acid) as sole carbon source has been isolated from the roots of tomato plants inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis. An intracellular esterase exhibiting very high affinity (K-m = 2 mu M) for chlorogenic acid has been extracted and purified by FPLC from the chlorogenate-grown cultures of this bacterium. The molecular mass of the purified esterase determined by SDS-PAGE was 61 kDa and its isoelectric point determined by chromatofocusing was 7.75. The esterase hydrolysed chlorogenic acid analogues (caffeoylshikimate, and the 4- and 3-caffeoylquinic acid isomers), feruloyl esterases substrates (methyl caffeate and methyl ferulate), and even caffeoyl-CoA in vitro but all of them were less active than chlorogenic acid, demonstrating that the esterase is a genuine chlorogenic acid esterase. It was also induced when the bacterial strain was cultured in the presence of hydroxycinnamic acids (caffeic, p-coumaric or ferulic acid) as sole carbon source, but not in the presence of simple phenolics such as catechol or protocatechuic acid, nor in the presence of organic acids such as succinic or quinic acids. The purified esterase was remarkably stable in the presence of methanol, rapid formation of methyl caffeate occurring when its activity was measured in aqueous solutions containing 10-60% methanol. Our results therefore show that this bacterial chlorogenase can catalyse the transesterification reaction previously detected during the methanolic extraction of chlorogenic acid from arbuscular mycorrhizal tomato roots. Data are presented suggesting that colonisation by Rhizophagus irregularis could increase chlorogenic acid exudation from tomato roots, especially in nutrient-deprived plants, and thus favour the growth of chlorogenate-metabolizing bacteria on the root surface or in the mycorhizosphere

Vers une standardisation des outils pour les études de protéomique clinique

L’identification de marqueurs protéiques d’intérêt diagnostique ou pronostique a suscité de grands espoirs en oncologie. À l’heure actuelle, cependant, la protéomique clinique fondée sur l’analyse de profils protéiques dans les liquides biologiques se heurte à un défaut de sensibilité et de reproductibilité. Cela est dû au manque de standardisation des étapes pré-analytiques et analytiques. La taille de la cohorte de patients analysés est un autre paramètre essentiel pour donner de la puissance à l’analyse envisagée. Le recrutement d’une cohorte suffisante justifie les études multicentriques qui imposent l’analyse de la reproductibilité inter-plates-formes et la standardisation des étapes pré-analytiques et analytiques. Cette standardisation passe par la mise en place d’échantillons de référence

On-chip detection, sizing and proteomics of extracellular vesicles

International audienceMicroparticles are small extracellular vesicles (EVs) (from ~100 to 1000 nm) produced by different cell types, through the budding of the plasma membrane, while exosomes (from ~30 to 120 nm) originate from the endolysosomal pathway before fusing with the plasma membrane to be released. Increased platelet-derived microparticles (PMPs) formation has been reported to contribute to the inflammatory role of blood components used for transfusion. When PMPs formation results from thrombin activation, they are able to aggregate monocyte cells in vitro. Nevertheless, the reason(s) for this EVs functionality/effect on target cells still need to be clarified, due to their high variety in size, protein composition and the potential concomitant presence of exosomes and small MPs in the analyzed samples

Comparison of 3 ionisation methods - electron ionisation, chemical ionisation and atmospheric pressure photoionisation for the characterisation of volatile organic compounds (VOCs)

International audienceGas chromatography (GC) is a reproducible, robust, selective and sensitive method to analyse volatile organic compounds (VOCs) in a wide range of applications. The separated analytes are generally characterised by mass spectrometry (MS) under vacuum conditions. The main ionisation method is the Electron Ionisation (EI): high energy exchanges occur, causing reproducible molecular fragmentations. Chemical Ionisation (CI) is another ionisation method where a reactive gas (i.e. methane or ammonia) is ionised to form reactant ions. GC-MS can also be conducted under atmospheric pressure. Atmospheric Pressure PhotoIonisation (APPI) is the most recent source [1]. Emitted photons give rise to quasi-molecular ions. In our research platform, we recently coupled a GC Trace 1310 to a High Resolution Mass Spectrometer (HRMS) Orbitrap Fusion (ThermoScientific) with the APPI source developed by Mascom (Bremen, Germany). In this work, first, we present a general overview of the technical developments carried out on 13 VOCs with the GC-APPI-HRMS hyphenated technique. Secondly, we compare the three ionisation methods listed above. For this purpose, we used 6 VOCs of different chemical classes to determine the Limit Of Detection (LOD) for each source. Positive detection in the Orbitrap allows a factor 2 to be gained in sensitivity. In MS2, High Collision Dissociation (HCD) is more informative than Collision-Induced Dissociation (CID). For the source parameters, it is better to decrease the sheath gas flow and the transfer tube temperature to 150°C in order to enhance the MS signal. The LOD reached for each VOC depends on the chemical class of the molecule. EI is the most sensitive method to characterise acids (0.03 ppm), alcohols (0.006 ppm), ketones and esters (0.003 ppm) while CI with methane as reactant gas is more appropriate for aldehydes (0.008 ppm). Even if GC-APPI-HRMS is not the most sensitive method (from 0.004 to 0.3 ppm), the LOD ranges found in our experiments is generally better than in the literature [2]. The developments in GC-APPI-HRMS are still in progress. (1) Reducing fragmentations and enhancing sensitivity by using acetone as a dopant gas, (2) conducting in vivo analyses, are our main objectives before transferring this very promising technology to aroma compounds characterisation in food extracts or in exhaled breath