The biological interpretation of metabolomic data can be misled by the extraction method used
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The field of metabolomics is getting more and more popular and a wide range of different sample preparation procedures are in use by different laboratories. Chemical extraction methods using one or more organic solvents as the extraction agent are the most commonly used approach to extract intracellular metabolites and generate metabolite profiles. Metabolite profiles are the scaffold supporting the biological interpretation in metabolomics. Therefore, we aimed to address the following fundamental question: can we obtain similar metabolomic results and, consequently, reach the same biological interpretation by using different protocols for extraction of intracellular metabolites? We have used four different methods for extraction of intracellular metabolites using four different microbial cell types (Gram negative bacterium, Gram positive bacterium, yeast, and a filamentous fungus). All the quenched samples were pooled together before extraction, and, therefore, they were identical. After extraction and GC–MS analysis of metabolites, we did not only detect different numbers of compounds depending on the extraction method used and regardless of the cell type tested, but we also obtained distinct metabolite levels for the compounds commonly detected by all methods (P-value < 0.001). These differences between methods resulted in contradictory biological interpretation regarding the activity of different metabolic pathways. Therefore, our results show that different solvent-based extraction methods can yield significantly different metabolite profiles, which impact substantially in the biological interpretation of metabolomics data. Thus, development of alternative extraction protocols and, most importantly, standardization of sample preparation methods for metabolomics should be seriously pursued by the scientific community.
KeywordsMetabolite extraction Metabolic fingerprint Metabolite profiling Metabolome Metabolomics Mass spectrometry
- Clark, W., & Christopher, K. (2000). An Introduction to DNA: Spectrophotometry, degradation, and the ‘Frankengel’ experiment. In S. J. Karcher (Ed.), Tested studies for laboratory reaching (Vol. 22, pp. 81–99). Edmonton, Canada: Association for Biology Laboratory Education, University of Alberta.Google Scholar
- Goldstone, D. C., Villas-Bôas, S. G., Till, M., Kelly, W. J., Attwood, G. T., & Arcus, V. L. (2009). Structural and functional characterization of a promiscuous feruloyl esterase (Est1E) from the rumen bacterium Butyrivibrio proteoclasticus. Proteins, 78, 1457–1469.Google Scholar
- Villas-Bôas, S. G., Koulman, A., & Lane, G. A. (2007). Method standardization. In J. Nielsen & M. C. Jewett (Eds.), Topics in current genetics: Metabolomics (Vol. 18, pp. 11–52). Heidelberg: Springer.Google Scholar
- Winder, C. L., Dunn, W. B., Schuler, S., Broadhurst, D., Jarvis, R., Stephens, G. M., et al. (2008). Global metabolic profiling of Escherichia coli cultures: An evaluation of methods for quenching and extraction of intracellular metabolites. Analytical Chemistry, 80, 2939–2948.PubMedCrossRefGoogle Scholar