Metabolism exploration platform (PFEM)

Scientific background

The aim of Clermont-Ferrand's Human Nutrition research is to improve human nutrition, particularly during aging. The study of metabolisms is of major interest, since it reflects the integration of all post-genomic phenomena. It is fundamental to understanding the functioning of living organisms at all levels of integration, from the cell to the ecosystem. This new high-throughput discipline makes it possible to tackle problems of biocomplexity by describing exhaustively the metabolites generated or modified under certain nutritional conditions. It involves the mass spectrometric analysis of samples containing complex matrices (biological fluids or tissues) and metabolites at low concentration levels, a tool developed by the platform.

The "Metabolism and Mass Spectrometry" team is a resource center for mass spectrometry and metabolomics within INRAE and is the main component of the "Exploration of Metabolism" Platform (PFEM). This platform, which received IBiSA accreditation in 2010, ISO9001 certification in 2008, and NFX50-900 certification in 2013, is a shared tool for studying the surrounding "metabolome," i.e., enhanced with proteomic and transcriptomic analysis capabilities. The team is also one of the four founding members of the MetaboHUB (national infrastructure for metabolomics and fluxomics) infrastructure of excellence.

As a component of a platform, the team is a cross-functional structure entirely dedicated to metabolic studies. It offers both analytical services and methodological developments. Its missions consist of :

• Provide skills and technical resources for the implementation of scientific projects
• Provide the expertise and advice necessary for the definition, implementation, execution, and management of specific projects and analyses
• Ensure technological monitoring, in connection with the development of new tools and protocols
• Provide technical assistance to users
• Offer initial and ongoing training, and conduct scientific outreach in the field.

Developed Analytical Approaches

The platform has extensive experience in the field of fluxomics. This discipline involves monitoring specific metabolites and determining their transfer rate between cellular (or rather tissue) compartments, which allows us to define their inter-organ flux. The platform's expertise in this field ranges from the quantification of small molecules present in different compartments of the body to measuring the isotopic enrichment of molecules, providing insight into their metabolic rate after administering these isotopes, used as biological tracers, to organisms.

The platform is also currently developing the use of the metabolomics approach. This new discipline, based on high-throughput analyses, makes it possible to address biocomplexity problems by exhaustively describing the metabolites generated or modified under the conditions studied. The skills currently offered by the platform in this area concern the determination of metabolic profiles by non-targeted multi-platform approaches in order to obtain maximum analytical coverage of the metabolome. In addition, they consist of the development of analytical strategies to facilitate the identification of metabolites (potential biomarkers), both through the use of very high resolution mass spectrometry and the creation of bioinformatics tools for automating data processing.

Equipment and Expertise

This center currently houses around ten complementary devices for mass spectrometry and isotope mass spectrometry. Six types of technologies are present on the platform :
• GC-C-IRMS: gas chromatography-combustion-isotope mass spectrometry dedicated to fluxomics (very low enrichment measurements): analyses of exhaled gas and amino acids incorporated into proteins.
• GC-MS and GC-QqQ: gas chromatography-single or triple quadrupole mass spectrometry, dedicated to quantification or fluxomics (free fatty acids and amino acids) and metabolomics (metabolic profiles of fecal water).
• LC-QqQ and QTrap: liquid chromatography-triple quadrupole or quadrupole ion trap mass spectrometry, dedicated to quantification or fluxomics: micronutrient (polyphenols, carotenoids, etc.) and amino acid analyses.
• LC-QToF and GC-QToF: liquid chromatography-quadrupole/time-of-flight mass spectrometry, dedicated to metabolomic activity: acquisition of urinary and plasma metabolic profiles.
• LTQ Orbitrap: very high-resolution spectrometer, dedicated to the identification of biomarkers during metabolomic studies.

The platform also has access to the following equipment:
• Metabolic Profler (LC/NMR/ToF): device dedicated to metabolomic studies (profiling, structure confirmation), installed at UCA (Université Clermont Auvergne, Clermont-Ferrand)
 

In-house research

Methodological developments relate to sample preparation, metabolite analysis, identification, and signal processing. In fluxomics, these primarily involve the quantification of isotopic enrichments using MS/MS approaches to increase the sensitivity and specificity of measurements. In metabolomics, developments involve optimizations and validations of analytical methods on various biological matrices, as well as the deployment of analytical and bioinformatics tools to optimize and automate data processing and biomarker identification.

Bioinformatics and Biostatistics Activities

In support of its analytical activities, the PFEM is developing expertise in BioAnalysis and Bioinformatics. The "Bioinformatics & Statistics" group focuses on preprocessing (filtering and cleaning data), descriptive and inferential data analyses, and the development of in silico identification and annotation strategies in the field of metabolomics. The PFEM's combined expertise in computer science, software development, statistics, and bioinformatics is behind projects such as PeakForest, an online library of metabolic profiles (peakforest.org) and enables active collaboration on various national and international projects (workflow4metabolomics, MetabOlism FramewOrk , base de données PhytoHUB).