11 research outputs found
Analyse du métabolome par chromatographie couplée à la spectrométrie de masse (application à la recherche de biomarqueurs et à la compréhension des systèmes biologiques)
Ce travail a pour objectif de développer des approches métabolomiques basées sur l utilisation de la spectrométrie de masse afin de les appliquer à des thématiques biologiques. Pour cela, des méthodes d analyse, de traitement des données, ainsi que d analyses statistiques multivariées ont ainsi été mises au point. Cette approche a été appliquée à l étude des effets du cadmium chez la plante Arabidopsis thaliana. des peptides chélatant ce métal qui n avaient jamais été décrits dans cette espèce ont ainsi été caractérisés. La régulation de la voie métabolique impliquée dans la synthèse de ces peptides a également été étudiée en détails. L analyse métabolomique a été appliquée à la recherche de biomarqueurs de fibrose pulmonaire sur des échantillons urinaires provenant de modèles murins. Enfin, des études ont également été menée afin d affiner nos stratégies d identification des métabolites d intérêt biologiques mis en évidence par les analyses statistiques multivariées.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF
La spectrométrie de masse pour l'analyse des biomolécules de petite taille dans les fluides biologiques
New insights into the regulation of phytochelatin biosynthesis in A. thaliana cells from metabolite profiling analyses.
In higher plants and some fungi, heavy metals induce the synthesis of chelating peptides known as phytochelatins (PCs). They are characterized by the general structure (gamma-Glu-Cys)(n)-Gly, but in some plant species, the C-terminal glycine can be replaced by serine, glutamine, glutamate or alanine, leading to iso-phytochelatins (iso-PCs). Although the distribution of iso-PCs is considered to differ from one species to another, we previously showed that Arabidopsis thaliana (A. thaliana) cells are able to synthesize most PC-related peptides (PCs and iso-PCs) described in the literature. We also observed an accumulation of the dipeptide gamma-glutamylcysteine (gamma-EC) when cadmium (Cd) (200 muM) was added to the culture medium, suggesting that either glutathione synthetase or glycine availability could be a limiting factor for the biosynthesis of PC-related peptides. In this context, the aim of the present work was to seek new insights into the regulation of PC synthesis by performing metabolic profiling using liquid chromatography-mass spectrometry. The levels of PC-related peptides and their precursors were measured in A. thaliana cells following Cd exposure. A range of doses (0, 50, 200 and 400 muM CdNO(3)) and kinetic studies (from 1 to 48 h) showed a dose threshold (50 muM CdNO(3)) and a lag time between the appearance of PCs and iso-PCs concomitant with the gamma-EC accumulation induced by Cd, occurring at cadmium concentrations above 50 muM. This accumulation was suppressed by supplementation of the culture medium with 25 mM glycine. Glycine supplementation had a limited impact on the concentrations of glutathione and PCs whereas the levels of most iso-PCs were significantly increased. Taken together, these results indicate that GSH is involved in the biosynthesis of the iso-PCs in vivo, and that the biosynthesis of PC-related peptides is limited by the availability of glycine in the presence of high cadmium concentrations
Metabolomic investigation of the response of the model plant Arabidopsis thaliana to cadmium exposure: Evaluation of data pretreatment methods for further statistical analyses.
International audienceUnderstanding the metabolic answer and the adaptation of plants towards heavy metal exposure opens the way to future phytoremediation of polluted sites. For this reason, we studied the impact of cadmium, a toxic heavy metal, on the metabolome of the model plant Arabidopsis thaliana. The analytical methodology (liquid chromatography coupled with mass spectrometry) that was used within the framework of a designed experiment conducted on A. thaliana cells exposed to cadmium generated an important volume of data. Multivariate statistical analyses appeared relevant to compare the metabolic fingerprints in order to isolate and identify some discriminating metabolites. Three types of data pretreatment, i.e., reduction of dimensionality, bucketing and automatic processing by the MetAlign™ software were compared for efficiency in extracting the information. The pretreated data were then subjected to multivariate statistical analysis by principal component analysis (PCA) and partial least square regression (PLS). Finally, an OSC (Orthogonal Signal Correction)-PLS2 approach performed on kinetic and dose ranging studies allowed to visualize time- and cadmium dose-induced changes on the metabolism of A. thaliana cells
Association of metabolomic and lipidomic approaches for plasma biomarkers discovery
<p>Metabolomics refers to the large scale detection and quantification of metabolites in a given biological condition. It is a multidisciplinary approach combining analytical chemistry, informatics and biostatistics. The comparison of metabolic profiles of biofluids from control and disease patients is currently used for biomarkers finding. However, these profiles obtained by liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) contain hundreds to thousands of features, most of them remaining unknown or at least not characterized in analytical systems, in particular in the field of biomarkers discovery. This makes necessary the use of robust data processing tools, i.e. peak detection and alignment algorithms and automatic annotation tools associated with public databases and an in-house metabolites (1200 metabolites to date) libraries.</p>
<p>The challenge is as well to obtain comprehensive untargeted metabolic profiles of biofluids with metabolites displaying huge differences in physicochemical properties, from polar metabolites to apolar lipids. In this respect, we have developed an analytical pipeline involving 3 orthogonal LC-HRMS systems for the analysis of polar to moderately apolar molecules. Different chromatographic retention mechanisms, i.e. reversed phase, hydrophilic interaction liquid chromatography (HILIC) and PentaFluroPhenylPropyl (PFPP) are thus performed on the same sample.</p>
<p>As a complementary approach, we have developed an analytical method for lipid profiling in plasma. This lipidomic method is based on dedicated sample preparation procedure, LC-HRMS conditions and data processing tools. When applied to human plasma samples, more than 900 unique lipid species, <em>i.e.</em> unique<em> m/z</em> – retention time signature, can be detected and identified, including glycerophospholipids, glycerolipids, shingolipids, free fatty acids and sterols and derivates.</p>
<p>This methodology was applied to the comparison of 101 plasma samples of patients suffering from spinocerebellar ataxia (SCA1, 2, 3 and 7), obtained from Neuromics partners (WP4). The first results on their lipid profiles analyses will be presented.</p
The early responses of Arabidopsis thaliana cells to cadmium exposure explored by protein and metabolite profiling analyses.
Development and validation of a multiresidue method for the analysis of more than 500 pesticides and drugs in water based on on-line and liquid chromatography coupled to high resolution mass spectrometry
Mass spectrometry for the identification of the discriminating signals from metabolomics: Current status and future trends
The metabolome is characterized by a large number of molecules exhibiting a high diversity of chemical structures and abundances, requiring complementary analytical platforms to reach its extensive Coverage. Among them, atmospheric pressure ionization mass spectrometry (API-MS)-based technologies, and especially those using electrospray ionization are now very popular. In this context. this review deals with strengths, limitations and future trends ill the identification of signals highlighted by API-MS-based metabolomics. It covers the identification process from the determination of the molecular mass and/or its elemental composition to the confirmation of structural hypotheses. Furthermore, some tools that were developed ill Order to address the MS signal redundancy and some approaches that could facilitate identification by improving the Visualization and organization of complex data sets are also reported and discussed. (C) 2008 Elsevier B.V. All rights reserved
High‐Resolution Mass Spectrometry Associated with Data Mining Tools for the Detection of Pollutants and Chemical Characterization of Honey Samples
International audienceno abstrac