Journal
ANALYTICAL BIOCHEMISTRY
Volume 451, Issue -, Pages 48-55Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2014.01.018
Keywords
Metabolomics; Gas chromatography-mass spectrometry; Escherichia coli; yfcC; Metabolic profiling
Funding
- National Basic Research Program of the State Ministry of Science and Technology of China [2007CB707800]
- Chinese Academy of Sciences
- National Natural Science Foundation of China [21175132, 21021004]
- National Science Foundation of Liaoning Province [2013020167]
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Metabolomics is a potent tool to assist in identifying the function of unknown genes through analysis of metabolite changes in the context of varied genetic backgrounds. However, the availability of a universal unbiased profiling analysis is still a big challenge. In this study, we report an optimized metabolic profiling method based on gas chromatography-mass spectrometry for Escherichia coli. It was found that physiological saline at -80 degrees C could ensure satisfied metabolic quenching with less metabolite leakage. A solution of methanol/water (21:79, v/v) was proved to be efficient for intracellular metabolite extraction. This method was applied to investigate the metabolome difference among wild-type E. coli, its yfcC deletion, and overexpression mutants. Statistical and bioinformatic analysis of the metabolic profiling data indicated that the expression of yfcC potentially affected the metabolism of glyoxylate shunt. This finding was further validated by real-time quantitative polymerase chain reactions showing that expression of aceA and aceB, the key genes in glyoxylate shunt, was upregulated by yfcC. This study exemplifies the robustness of the proposed metabolic profiling analysis strategy and its potential roles in investigating unknown gene functions in view of metabolome difference. (C) 2014 Elsevier Inc. All rights reserved.
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