title: Analytical methodology for metabolomics study of adherent mammalian cells using NMR, GC-MS and LC-HRMS.
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.
authors.affiliation: INSERM U930 "Imagerie et Cerveau", CHRU de Tours, Université François-Rabelais, 10 Bv Tonnellé, 37044, Tours, France.
authors.forename: Blandine
authors.initials: B
authors.lastname: Madji Hounoum
authors.affiliation: INSERM U930 "Imagerie et Cerveau", CHRU de Tours, Université François-Rabelais, 10 Bv Tonnellé, 37044, Tours, France.
authors.forename: Hélène
authors.initials: H
authors.lastname: Blasco
authors.affiliation: INSERM U930 "Imagerie et Cerveau", CHRU de Tours, Université François-Rabelais, 10 Bv Tonnellé, 37044, Tours, France.
authors.forename: Lydie
authors.initials: L
authors.lastname: Nadal-Desbarats
authors.affiliation: INSERM U930 "Imagerie et Cerveau", CHRU de Tours, Université François-Rabelais, 10 Bv Tonnellé, 37044, Tours, France.
authors.forename: Binta
authors.initials: B
authors.lastname: Diémé
authors.affiliation: INSERM U930 "Imagerie et Cerveau", CHRU de Tours, Université François-Rabelais, 10 Bv Tonnellé, 37044, Tours, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Montigny
authors.affiliation: INSERM U930 "Imagerie et Cerveau", CHRU de Tours, Université François-Rabelais, 10 Bv Tonnellé, 37044, Tours, France.
authors.forename: Christian R
authors.initials: CR
authors.lastname: Andres
authors.affiliation: INSERM U930 "Imagerie et Cerveau", CHRU de Tours, Université François-Rabelais, 10 Bv Tonnellé, 37044, Tours, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Emond
authors.affiliation: INSERM U930 "Imagerie et Cerveau", CHRU de Tours, Université François-Rabelais, 10 Bv Tonnellé, 37044, Tours, France. mavel@univ-tours.fr.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Mavel
mesh_terms: D000818:Animals; D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D055432:Metabolomics / Q000379:methods*; D051379:Mice; D015203:Reproducibility of Results; D059010:Single-Cell Analysis / Q000379:methods*
keywords: Bioanalytical methods; Cell systems/single cell analysis; Mass spectrometry/ICP-MS; NMR/ESR