Abstract
The comprehensive metabolomic analyses using eukaryotic and prokaryotic cells are an effective way to identify biomarkers or biochemical pathways which can then be used to characterize disease states, differences between cell lines or inducers of cellular stress responses. One of the most commonly used extraction methods for comprehensive metabolomics is the Bligh and Dyer method (BD) which separates the metabolome into polar and nonpolar fractions. These fractions are then typically analysed separately using hydrophilic interaction liquid chromatography (HILIC) and reversed-phase (RP) liquid chromatography (LC), respectively. However, this method has low sample throughput and can also be biased to either polar or nonpolar metabolites. Here, we introduce a MeOH/EtOH/H2O extraction paired with HILIC-time-of-flight (TOF)-mass spectrometry (MS) for comprehensive and simultaneous detection of both polar and nonpolar metabolites that is compatible for a wide array of cellular species cultured in different growth media. This method has been shown to be capable of separating polar metabolites by a HILIC mechanism and classes of lipids by an adsorption-like mechanism. Furthermore, this method is scalable and offers a substantial increase in sample throughput compared to BD with comparable extraction efficiency. This method was able to cover 92.2 % of the detectable metabolome of Gram-negative bacterium Sinorhizobium meliloti, as compared to 91.6 % of the metabolome by a combination of BD polar (59.4 %) and BD nonpolar (53.9 %) fractions. This single-extraction HILIC approach was successfully used to characterize the endometabolism of Gram-negative and Gram-positive bacteria as well as mammalian macrophages. FigureThe extraction and ionization efficiency of MeOH/EtOH/H2O HILIC approach encompasses both the polar and nonpolar fractions from Bligh and Dyer extraction Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-014-7797-5) contains supplementary material, which is available to authorized users.
Highlights
Cellular metabolomics is an important part of systems biology as it reflects the phenotype of cells and monitors cellular activities in a perturbed system [1, 2]
Bligh and Dyer method (BD) extraction is commonly used for comprehensive metabolomics to ensure full coverage of both polar and nonpolar metabolites by running both polar and nonpolar phases separately on hydrophilic interaction liquid chromatography (HILIC) and RPLC
The scalable extraction method was tested on a 100-μL S. meliloti (2×109) cell culture grown in M9 growth medium in a 96-well microtiter plate
Summary
Cellular metabolomics is an important part of systems biology as it reflects the phenotype of cells and monitors cellular activities in a perturbed system [1, 2]. The overall metabolome coverage observed with this single-extraction HILIC approach is equivalent to the BD method with separate analyses of polar and nonpolar fractions. An extraction solvent of 2:2:1 MeOH/EtOH/H2O or 1:1 MeOH/EtOH was used to generate a single fraction which contained a mixture of polar and nonpolar lipid metabolites.
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