Comprehensive and simultaneous coverage of lipid and polar metabolites for endogenous cellular metabolomics using HILIC-TOF-MS

• Published in: Analytical and bioanalytical chemistry Vol. 406; no. 15; pp. 3723 - 3733
• Main Authors: Fei, Fan, Bowdish, Dawn M. E., McCarry, Brian E.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.06.2014; Springer; Springer Nature B.V
• Subjects: Adsorption; Algorithms; Analytical Chemistry; Arrays; Bacteria; biochemical pathways; Biochemistry; biomarkers; Biomarkers - analysis; Cells, Cultured; Cellular; Characterization and Evaluation of Materials; Chemical properties; Chemistry; Chemistry and Materials Science; Chromatography; Chromatography, High Pressure Liquid; Chromatography, Liquid; Chromatography, Reverse-Phase; culture media; Ensifer meliloti; Extraction; Food Science; Gram-negative bacteria; Gram-positive bacteria; Growth media; High performance liquid chromatography; hydrophilic interaction chromatography; Hydrophobic and Hydrophilic Interactions; Hypotheses; Identification and classification; Ions; Laboratory Medicine; Lipids; Lipids - chemistry; Liquid chromatography; macrophages; Macrophages - cytology; Macrophages - drug effects; mammals; Mass Spectrometry; Metabolism; Metabolites; Metabolome; Metabolomics; methanol; Methods; Monitoring/Environmental Analysis; prokaryotic cells; Reproducibility of Results; Research Paper; Scientific imaging; Sinorhizobium; Sinorhizobium meliloti; Solvents - chemistry; spectroscopy; stress response; Time-of-flight mass spectrometry; Toxicology; Water - chemistry; Canada; Lipids; Bacteria; Comprehensive metabolomics; Polar metabolites; HILIC-TOF-MS; Macrophage
• ISSN: 1618-2642; 1618-2650; 1618-2650
• DOI: 10.1007/s00216-014-7797-5

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/H 2 O 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. Figure The extraction and ionization efficiency of MeOH/EtOH/H2O HILIC approach encompasses both the polar and nonpolar fractions from Bligh and Dyer extraction