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

被引:63
作者
Fei, Fan [1 ,2 ]
Bowdish, Dawn M. E. [2 ]
McCarry, Brian E. [1 ]
机构
[1] McMaster Univ, Dept Chem & Chem Biol, Hamilton, ON L8S 4M1, Canada
[2] McMaster Univ, Dept Pathol & Mol Med, Michael G DeGroote Inst Infect Dis Res, Hamilton, ON L8N 3Z5, Canada
基金
加拿大自然科学与工程研究理事会;
关键词
Comprehensive metabolomics; Polar metabolites; Lipids; HILIC-TOF-MS; Bacteria; Macrophage; HYDROPHILIC INTERACTION CHROMATOGRAPHY; MASS-SPECTROMETRY; SAMPLE PREPARATION; PHOSPHOLIPID CLASSES; SEPARATION; COMBINATION; EXTRACTION; STRATEGIES;
D O I
10.1007/s00216-014-7797-5
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:3723 / 3733
页数:11
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