Global metabolic profiling of Escherichia coli cultures: an evaluation of methods for quenching and extraction of intracellular metabolites
| Autor: | Roger M. Jarvis, Stephanie Schuler, Royston Goodacre, Gillian M. Stephens, Catherine L. Winder, David Broadhurst, Warwick B. Dunn |
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| Rok vydání: | 2008 |
| Předmět: |
Bacteriological Techniques
Chromatography Metabolite Systems Biology Chemostat Mass spectrometry Gas Chromatography-Mass Spectrometry Analytical Chemistry chemistry.chemical_compound Metabolomics Metabolism Biochemistry chemistry Metabolome Escherichia coli Sample preparation Solid phase extraction Gas chromatography |
| Zdroj: | Analytical chemistry. 80(8) |
| ISSN: | 1520-6882 |
| Popis: | Metabolomics and systems biology require the acquisition of reproducible, robust, reliable, and homogeneous biological data sets. Therefore, we developed and validated standard operating procedures (SOPs) for quenching and efficient extraction of metabolites from Escherichia coli to determine the best methods to approach global analysis of the metabolome. E. coli was grown in chemostat culture so that cellular metabolism could be held in reproducible, steady-state conditions under a range of precisely defined growth conditions, thus enabling sufficient replication of samples. The metabolome profiles were generated using gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS). We employed univariate and multivariate statistical analyses to determine the most suitable method. This investigation indicates that 60% cold (-48 degrees C) methanol solution is the most appropriate method to quench metabolism, and we recommend 100% methanol, also at -48 degrees C, with multiple freeze-thaw cycles for the extraction of metabolites. However, complementary extractions would be necessary for coverage of the entire complement of metabolites as detected by GC/TOF-MS. Finally, the observation that metabolite leakage was significant and measurable whichever quenching method is used indicates that methods should be incorporated into the experiment to facilitate the accurate quantification of intracellular metabolites. |
| Databáze: | OpenAIRE |
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