Journal
ANALYTICAL CHEMISTRY
Volume 85, Issue 4, Pages 2169-2176Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac302881e
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Funding
- Pioneer Research Center Program [2011-0002327]
- Advanced Biomass R&D Center of Korea [2011-0031353]
- Korean Government (MEST)
- Direct For Biological Sciences [1153491] Funding Source: National Science Foundation
- Div Of Molecular and Cellular Bioscience [1153491] Funding Source: National Science Foundation
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Metabolome sampling is one of the most important factors that determine the quality of metabolomics data. The main steps in metabolite sample preparation include quenching and metabolite extraction. Quenching with 60% (v/v) cold methanol at -40 degrees C has been most commonly used for Saccharomyces cerevisiae, and this method was recently modified as leakage-free cold methanol quenching using pure methanol at -40 degrees C. Boiling ethanol (75%, v/v) and cold pure methanol are the most widely used extraction solvents for S. cerevisiae. In the present study, metabolome sampling protocols, including the above methods, were evaluated by analyzing 110 identified intracellular metabolites of S. cerevisiae using gas chromatography/time-of-flight mass spectrometry. According to our results, fast filtration followed by washing with an appropriate volume of water can minimize the metabolite loss due to cell leakage as well as the contamination by extracellular metabolites, For metabolite extraction, acetonitrile/water mixture (1:1, v/v) at -20 degrees C was the most effective. These results imply that the systematic evaluation of existing methods and the development of customized methods for each microorganism are critical for metabolome sample preparation to facilitate the reliable and accurate analysis of metabolome.
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