Evaluation and optimization of sample preparation methods for metabolic profiling analysis of Escherichia coli.

Abstract

In this study, exemplified by Escherichia coli, a sample preparation method was developed and evaluated with more concerns on cell pellets washing, disruption, and metabolite extraction for global metabolite profiling analysis using GC-MS. We found that loss of some intracellular metabolites occurred inevitably, no matter which washing strategy was used. But PBS washing resulted in the least leakage of intercellular metabolites among the six tested methods. In addition, bead-beating showed better repeatability and high throughput than sonication for cell pellet disruption. Total six extraction solvents, including chloroform/methanol/water (2:0.21:0.79, v/v/v), methyl tert-butyl ether/methanol/water (2:0.21:0.79), acetonitrile/methanol/water (2:2:1), water/isopropanol/methanol (3:2:5), pure methanol, and methanol/water (1:1), were evaluated for metabolite extraction efficiency. We demonstrated that the numbers of extracted metabolites were almost the same, but methyl tert-butyl ether/methanol/water and chloroform/methanol/water could achieve the best recoveries in view of MS intensities. This study implies the necessity to evaluate the potential effects of serial sample preparation procedures prior to metabolomic analysis.