Evaluation of sampling and extraction methodologies for the global metabolic profiling of Saccharophagus degradans

Metabolomics is based on the unbiased and global analysis of metabolites of organisms at specific time points. Therefore, the nonselective and reproducible recovery of all existing metabolites while preventing their transformation is the ideal criterion for metabolome sample preparation. We evaluated currently used sampling methods and extraction solvents for global metabolic profiling of a gram-negative bacterium, Saccharophagus degradans, using gas chromatography-time-of-flight mass spectrometry (GC-TOF MS) with an emphasis on three steps: the sampling method, which consisted of cold methanol quenching or fast filtration; the subsequent washing step; and the extraction solvents. After cold methanol quenching with 70% (v/v) methanol at -70 degrees C, washing with 2.3% NaCl produced a serious loss of intracellular metabolites. In addition, when cold methanol quenching and fast filtration were compared, severe cell leakage caused by cold methanol quenching resulted in a significant loss of intracellular metabolites, which was confirmed by spectrometric analysis at 260 and 280 nm. Upon evaluation of extraction solvents such as pure methanol (MeOH), acetonitrile/water (50ACN; 1:1, v/v), acetonitrile/methanol/water mixture (AMW; 2:2:1), and water/isopropanol/methanol (WiPM; 2:2:5). AMW and WiPM were found to be superior extraction solvents for S. degradans based on the total peak intensities of the metabolites, the total number of metabolite peaks, and the reproducibility of recovered metabolite quantities; however, the metabolite profiles differed significantly between AMW and WiPM. This is the first evaluation of each step of sample preparation involved in global scale metabolic analysis by GC-TOF MS, which can be used as a model in the preparation of organism-specific samples for metabolome analysis.