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Analytical methodology for metabolomics study of adherent mammalian cells using NMR, GC-MS and LC-HRMS

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Abstract

We developed a methodology for the analysis of intracellular metabolites using nuclear magnetic resonance spectrometry (NMR), gas-chromatography coupled with mass spectrometry (GC-MS), and liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS). The main steps for analysis of adherent cells in order to recover the widest possible range of intracellular compounds are blocking metabolic activity by quenching and extraction of intracellular metabolites. We explored three protocols to quench NSC-34 cell metabolism and four different extraction methods, analyzed by NMR. On the basis of the number of metabolites extracted and their relative standard deviation (RSD) analyzed by NMR, the most reproducible protocol [quenching by MeOH at −40 °C and extraction with CH2Cl2/MeOH/H2O (3:3:2)] was used to obtain intracellular media to be analyzed by GC-MS and LC-HRMS. GC-MS analysis was optimized by three oximation procedures followed by silylation derivatization and these were compared to silylation alone. Using reversed-phase liquid chromatography (C18), four different gradients for LC-MS were compared. The analytical protocols were determined to establish the reliability and suitability of sample treatments required to achieve the correct biological analysis of untargeted mammalian cell metabolomics.

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Acknowledgments

We thank the “Département d’Analyses Chimiques et S.R.M. Biologique et Médicale” (PPF, Tours, France) for chemical analyses and especially Cinzia Bocca for LC-HRMS analyses.

Funding

This study was funded by ARSLA (“Association pour la Recherche sur la Sclérose Latérale Amyotrophique et autres maladies du motoneurone”) with a research grant and by “La Région Centre” with a PhD graduate grant. This work was supported by the “Institut National de la Santé et de la Recherche” INSERM and the University François-Rabelais.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. INSERM U930 “Imagerie et Cerveau”, CHRU de Tours, Université François-Rabelais, 10 Bv Tonnellé, 37044, Tours, France

    Blandine Madji Hounoum, Hélène Blasco, Lydie Nadal-Desbarats, Binta Diémé, Frédéric Montigny, Christian R. Andres, Patrick Emond & Sylvie Mavel

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  1. Blandine Madji Hounoum
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  2. Hélène Blasco
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  3. Lydie Nadal-Desbarats
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  4. Binta Diémé
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  5. Frédéric Montigny
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  6. Christian R. Andres
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  7. Patrick Emond
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  8. Sylvie Mavel
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Correspondence to Sylvie Mavel.

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Madji Hounoum, B., Blasco, H., Nadal-Desbarats, L. et al. Analytical methodology for metabolomics study of adherent mammalian cells using NMR, GC-MS and LC-HRMS. Anal Bioanal Chem 407, 8861–8872 (2015). https://doi.org/10.1007/s00216-015-9047-x

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