Strategy for choosing extraction procedures for NMR-based metabolomic analysis of mammalian cells
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Metabolomic analysis of mammalian cells can be applied across multiple fields including medicine and toxicology. It requires the acquisition of reproducible, robust, reliable, and homogeneous biological data sets. Particular attention must be paid to the efficiency and reliability of the extraction procedure. Even though a number of recent studies have dealt with optimizing a particular protocol for specific matrices and analytical techniques, there is no universal method to allow the detection of the entire cellular metabolome. Here, we present a strategy for choosing extraction procedures from adherent mammalian cells for the global NMR analysis of the metabolome. After the quenching of cells, intracellular metabolites are extracted from the cells using one of the following solvent systems of varying polarities: perchloric acid, acetonitrile/water, methanol, methanol/water, and methanol/chloroform/water. The hydrophilic metabolite profiles are analysed using 1H nuclear magnetic resonance (NMR) spectroscopy. We propose an original geometric representation of metabolites reflecting the efficiency of extraction methods. In the case of NMR-based analysis of mammalian cells, this methodology demonstrates that a higher portion of intracellular metabolites are extracted by using methanol or methanol/chloroform/water. The preferred method is evaluated in terms of biological variability for studying metabolic changes caused by the phenotype of four different human breast cancer cell lines, showing that the selected extraction procedure is a promising tool for metabolomic and metabonomic studies of mammalian cells. The strategy proposed in this paper to compare extraction procedures is applicable to NMR-based metabolomic studies of various systems.
KeywordsMetabolomics NMR Human breast cancer Metabolite extraction Sample preparation
This work was financed in part by a grant from the French Ministry of Research. We would like to thank Pr. Vehary Sakanyan, Dr. Garabet Yeretssian, and Dr. Sophie Barille-Nion for providing human breast cancer cell lines and for helpful discussions. We also acknowledge Michel Giraudeau for linguistic assistance. This research was supported in part by funding from the "Agence Nationale de la Recherche" for young researchers (ANR Grant 2010-JCJC-0804-01).
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