Dimanche 25 Octobre 2020                           


EBSI Team | Metabolimics

8. Multi-dimensional NMR metabolomics


Current team members:Aurore Michaud, Estelle Martineau, Jonathan Farjon, Serge Akoka, Patrick Giraudeau*

Past members: Adrien le guennec, Jérémy Marchand

Collaborations (past and present): Catherine Deborde and Annick Moing (INRA Bordeaux), Gaud Dervilly-Pinel and Yann Guitton (LABERCA Nantes), Olivier Grovel and Yves-François Pouchus (MMS Nantes), Jean-Charles Portais (INSA Toulouse)
Workflow for high-throughput targeted quantitative metabolomics by fast 2D NMR

While NMR-based metabolomics studies are mostly performed with 1H 1D NMR, the latter suffers from the severe and numerous peak overlaps characterizing complex biological samples. In this context, we are evaluating the potential of various 2D NMR approaches for a variety of targeted and untargeted metabolomics applications. Fast and repeatable 2D NMR methods are indispensable in this field, not only for the sake of high-throughput analysis, but also to reduce the impact of hardware instabilities and consequently improve the precision.

Multi-dimensional methods based on ultrafast 2D NMR or non-uniform sampling have already been applied in different fields through past and current collaborations. These applications include the targeted quantification of polar metabolites in tomato extracts at different development stages, or the untargeted lipidomics analysis of the effect of a growth promoter in meat. A similar strategy has been applied on a benchtop spectrometer, showing that the authentication of food products through untargeted strategies can be significantly improved by incorporating fast 2D NMR methods in the analytical workflow.

References:
  • P. Giraudeau, S. Massou, Y. Robin, E. Cahoreau, J.-C. Portais, S. Akoka, Ultrafast Quantitative 2D NMR: An Efficient Tool for the Measurement of Specific Isotopic Enrichments in Complex Biological Mixtures, Anal. Chem. 83, 3112 (2011).
  • A. Le Guennec, P. Giraudeau, S. Caldarelli, Evaluation of Fast 2D NMR for Metabolomics, Anal. Chem. 86, 5946 (2014).
  • T. Jézéquel, C. Deborde, M. Maucourt, V. Zhendre, A. Moing, P. Giraudeau, Absolute quantification of metabolites in tomato fruit extracts by fast 2D NMR, Metabolomics 11, 1231 (2015).
  • J. Marchand, E. Martineau, Y. Guitton, G. Dervilly-Pinel, P. Giraudeau, Multidimensional NMR approaches towards highly resolved, sensitive and high-throughput quantitative metabolomics, Curr. Op. Biotechnol. 43, 49 (2017).
  • J. Marchand, E. Martineau, Y. Guitton, B. Le Bizec, G. Dervilly-Pinel, P. Giraudeau, A multidimensional 1H NMR lipidomics workflow to address chemical food safety issues, Metabolomics 14, 60, (2018)
  • P. Giraudeau, NMR-based metabolomics and fluxomics: developments and future prospects, Analyst, in press (2020) : view online


  • Supported by: Région Pays de la Loire (RFI Food 4.2 Lipidotool), Biogenouest (CORSAIRE platform)

    9. Multiscale NMR for investigating lipidic metabolism of microalgae


    Current team members: Dylan Bouillaud, Benoît Charrier, Patrick Giraudeau, Jonathan Farjon*

    Collaborations (past and present): Olivier Gonçalves, Jérémy Pruvost and Jack Legrand (GEPEA, Univ. Nantes)


    Microalgae are photosynthetic organisms with a very high biochemical diversity and a great ability to produce valuable biomass. Only a minor part of the millions of species living on earth are currently known, therefore microalgae form a subject with a strong investigation potential. Some microalgae such as Parachlorella Kessleri and Nannochloropsis Gaditana are known to accumulate lipids under nitrogen starving stress conditions. This behavior is interesting because it forms the basis of the third generation biofuel production, a promising alternative energy.

    Microalgae are usually detected by cytometry at the cell scale, or by Infra-red and Raman spectroscopies but these techniques fail to detect intra-cellular chemicals due to the signal of the culture water.

    In this context, we are exploring the potential of multiscale NMR for the analysis of microalgae cells and their lipidic extracts. The main challenge is to identify and quantify lipids in microalgae aqueous cultures. With this aim, high field NMR provides the best sensitivity and resolution to decipher complex lipidic extracts. Moreover, benchtop NMR has a great ability for real time online monitoring of bioprocesses such as the accumulation of lipids in microalgae during a nitrogen starvation in photobioreactor. Recent improvements in LF NMR pulse sequences, especially in solvent suppression and ultra-resolved NMR are promising for profiling the lipidic metabolism of microalgae.

    Key references
  • D. Bouillaud, J. Farjon, O. Gonçalves, P. Giraudeau, Benchtop NMR for the monitoring of bioprocesses, Magn. Reson. Chem. in press (2019)
  • B. Gouilleux, S. Akoka, P. Giraudeau, Gradient-based solvent suppression methods on a benchtop spectrometer, Magn. Reson. Chem. 55, 91 (2017)
  • D. Bouillaud, V. Heredia, T. Castaing-Cordier, D. Drouin, B. Charrier, O. Gonçalves, J. Farjon, P. Giraudeau, Benchtop flow NMR spectroscopy as an online device for the in vivo monitoring of lipid accumulation in microalgae, Algal Res. 43, 101624 (2019)

  • Supported by Région Pays de la Loire (Pari scientifique AMER-METAL), CNRS (Projet interdisciplinarité RMN-(ME)2-TAL).

    10. Profiling by NMR


    Current team members: Lenny Haddad, Vincent Portaluri, Virginie Silvestre, Mathilde Grand, Patrick Giraudeau, Serge Akoka, Gérald Remaud*.

    Past team members: Ghina Hajjar, Denis Loquet, Boris Gouyeux, Sophie Guyader, Noëlle Merchack.

    Collaborations: J. Bejjani, T. Rizk (Saint-Joseph University, Beirut, Lebanon), F. Thomas, E. Jamin (EUROFINS, Nantes, France),


    Classification of olive oils according to the geographical origin

    We apply high-field NMR combined with advanced statistical analysis methods for the profiling of complex samples (biological fluids, extracts, foodstuffs, etc.). This profiling approach forms part of our “omics” expertise as a member of the CORSAIRE metabolomics core facility (part of the Biogenouest network). Profiling tools include 1D 1H and 13C NMR, but also innovative fast 2D NMR methods. These approaches are applied both at high-field and on bencthop NMR spectrometers.

    Beyond the classical ”omics” workflow, particularly original work concerns the development of profiling methods for lipid mixtures as found in edible fats. Such mixtures may be authenticated by the metabolic profile but also by the determination of the isotopic profile of major components. We show that a combined 13C-isotopomics and metabolomics approach presents many advantages: no need for prior chemical manipulations, determination of the positional distribution of major fatty acids on the glycerol backbone of triglycerides, and measurement of the relative position-specific isotopic 13C content at different chemical sites.

    Furthermore, all this information is obtained from the same spectrum in a few minutes. This method has been compared with 1H profiling and gas chromatography. Its higher efficiency is explained by a significant contribution of variables that can only be obtained by this technique. Generally speaking, 13C profiling enables the classification of all types of edible fats.

    References:
  • N. Merchak, V. Silvestre, L. Rouger, P. Giraudeau, T. Rizk, J. Bejjani, S. Akoka. Precise and rapid isotopomic analysis by 1H-13C HSQC: Application to triacylglycerol matrices. Talanta, 2016, 156-157, 239-244.
  • N. Merchak, E. El Bacha, T. Rizk, S. Akoka, J. Bejjani. Geoclimatic, varietal, and temporal effects on the Lebanese olive oils composition and classification: A 1H NMR metabolomic study. Food Chemistry, 2017, 217, 379-388.
  • N. Merchak, V. Silvestre, D. Loquet, T. Rizk, S. Akoka, J. Bejjani. A strategy for simultaneous determination of fatty acid composition, fatty acid position, and position-specific isotope contents in triacylglycerol matrices by C-13-NMR. Analytical and Bioanalytical Chemistry, 2017, 409(1), 307-3015.
  • N. Merchak, T. Rizk, V. Silvestre, G.S. Remaud, J. Bejjani, S. Akoka. Olive oil characterization and classification by 13C-NMR with a polarization transfer technique: A comparison with gas chromatography and 1H-NMR. Food Chemistry, 2017, 245, 717-723.
  • S. Guyader, F. Thomas, V. Portaluri, E. Jamin, S. Akoka, V. Silvestre, GS Remaud. Authentication of edible fats and oils by non-targeted C-13 INEPT NMR spectroscopy. Food Control 91, 216-224 (2018).

  • Supported by: National Council for Scientific research of Lebanon, Saint-Joseph University of Beirut, EUROFINS Nantes, National Council for Scientific research and University of Nantes.