Strategy for choosing extraction procedures for NMR-based metabolomic analysis of mammalian cells

Abstract

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 (1)H 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.

Metabolic responses induced by DNA damage and poly (ADP-ribose) polymerase (PARP) inhibition in MCF-7 cells

“…The cells were then detached using a cell scraper and pipetted into 50 ml centrifuge tube. A dual phase extraction procedure adopted from Martineau et al (Martineau et al 2011) was used to extract the intracellular metabolites. Briefly, the quenched cells suspended in a mixture of chloroform, methanol and water in the ratio of 6:6:5.4 to make the final volume of 17.4 ml.…”

Analysis of Bacterial Biofilms Using NMR-Based Metabolomics

“…The NMR resonances from the biomolecules rapidly decay during the CPMG pulse. Alternatively, proteins and other biomolecules can be removed by an appropriate choice of extraction solvents [188,189]. The large interfering signal from water or other buffer components is also eliminated by the use of appropriate NMR solvent-suppression methods and a 100% deuterated buffer [190].…”

Metabolomics Identifies Metabolic Markers of Maturation in Human Pluripotent Stem Cell-Derived Cardiomyocytes

“…The cells were then detached using a cell scraper and pipetted into a 50 ml centrifuge tube. A dual phase extraction procedure adopted from Martineau et al 17 was used to extract the intracellular metabolites. Briefly, the quenched cells were suspended in a mixture of chloroform, methanol and water in the ratio of 6:6:5.4 to achieve a final volume of 17.4 ml.…”