Monday, November 9, 2009
P40

Corroboration of quantitative microbial metabolomics data by NET-analysis

Karin M. Overkamp1, Stefan J. Jol2, Bas Muilwijk1, Richard Bas1, Leon Coulier1, Matthias Heinemann2, and Mariët J. van der Werf1. (1) Microbiology, TNO, P.O. Box 360, Zeist, 3700AJ, Netherlands, (2) Institute of Molecular Systems Biology, ETH Zurich, Wolfgang-Pauli-Strasse 16, Zurich, 8093, Switzerland

Metabolomics focuses on the quantification in a single experiment of all metabolites present within a cellular extract. As the metabolomics-protocol involves many critical steps [1], an overall validation tool to check the quality of the reported results would be a great help in determining the usefulness of the metabolomics-experiment. In this study Network-Embedded Thermodynamic analysis [2] (NET-analysis) is employed as such a validation tool. NET-analysis combines thermodynamic laws with the known metabolic network operation in the cell and checks whether the intracellular concentrations as quantified by metabolomics are thermodynamically feasible. In addition, it yields insight into which reactions might be actively regulated by the cell and it can be used to infer flux directions of reactions for which the operational direction is unknown.

An interesting biological phenomenon, which also provides the NET-analysis with a set of different metabolomes, is the diauxic shift of E. coli after growth on glucose to consumption of previously produced but toxic acetate. We cultivated E. coli in controlled batchculture on glucose, took samples at different time points in the growth curve, quenched the metabolism and subsequently analyzed the cellular extracts by GC-MS and LC-MS techniques [1]. The measured data were converted into intracellular concentrations and for each individual timepoint in the growth curve a NET-analysis [2] was performed. Preliminary results indicate that for glucose catabolism the metabolome data were thermodynamically feasible.

[1] M.J. van der Werf et al., (2007) Analytical Biochemistry, Vol 370, pp 17-25
[2] A. Kümmel et al., (2006) Molecular Systems Biology doi:10.1038/msb4100074


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