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Metabolomics desire the gain of reproducible, reliable, and homogeneous biological data sets. Intracellular metabolite is a reflection of all the metabolic functions of an organism under any particular growth condition. For the development of experimental procedure, extraction of the metabolome to quantitative analysis of the metabolites is required. Klebsiella oxytoca was grown in chemostat culture so that cellular metabolism could be held in reproducible, steady-state conditions under a range of defined growth conditions, thus enabling sufficient replication of samples. In the absence of in situ methods capable of universally measuring metabolite pools, intracellular metabolite measurements need to be performed in vitro after extraction. This study indicates that 60% cold (-48 °C) methanol solution is the most appropriate method to quench metabolism. In this study, metabolites were extracted from Klebsiella oxytoca using six different commonly used procedures including acid or alkaline treatments, high-temperature extraction in the presence of ethanol or methanol or water and by lysis with chloroform-methanol. Intracellular metabolites were extracted by the six methods from cells grown under identical conditions. The intracellular metabolite profiles were generated using HPLC system coupled with a mass spectrometer.