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The biological interpretation of metabolomic data can be misled by the extraction method used

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Abstract

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.

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References

  • Aggio, R. B. M., Ruggiero, K., & Villas-Bôas, S. G. (2010). Pathway Activity Profiling (PAPi): From the metabolite profile to the metabolic pathway activity. Bioinformatics, 26, 2969–2976.

    Article  PubMed  CAS  Google Scholar 

  • Asiago, V. M., Alvarado, L. Z., Shanaiah, N., Gowda, G. A. N., Owusu-Sarfo, K., Ballas, R. A., et al. (2010). Early detection of recurrent breast cancer using metabolite profiling. Cancer Research, 70, 8309–8318.

    Article  PubMed  CAS  Google Scholar 

  • Baker, M. (2011). Metabolomics: From small molecules to big ideas. Nature Methods, 8, 117–121.

    Article  CAS  Google Scholar 

  • Bennett, B. D., Yuan, J., Kimball, E. H., & Rabinowitz, J. D. (2008). Absolute quantitation of intracellular metabolite concentrations by an isotope ratio-based approach. Nature Protocols, 3, 1299–1311.

    Article  PubMed  CAS  Google Scholar 

  • Canelas, A. B., Pierick, A. T., Ras, C., Seifar, R. M., van Dam, J. C., van Gulik, W. M., et al. (2009). Quantitative evaluation of intracellular metabolite extraction techniques for yeast metabolomics. Analytical Chemistry, 81, 7379–7389.

    Article  PubMed  CAS  Google Scholar 

  • 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.

  • Dietmair, S., Timmins, N. E., Gray, P. P., Nielsen, L. K., & Kromer, J. O. (2010). Towards quantitative metabolomics of mammalian cells: Development of a metabolite extraction protocol. Analytical Biochemistry, 404, 155–164.

    Article  PubMed  CAS  Google Scholar 

  • Dunn, W. B., Broadhurst, D. I., Atherton, H. J., Goodacre, R., & Griffin, J. L. (2011). Systems level studies of mammalian metabolomes: The roles of mass spectrometry and nuclear magnetic resonance spectroscopy. Chemical Society Reviews, 40, 387–426.

    Article  PubMed  CAS  Google Scholar 

  • El Rammouz, R., Letisse, F., & Durand, S. (2010). Analysis of skeletal muscle metabolome: Evaluation of extraction methods for targeted metabolite quantification using liquid chromatography tandem mass spectrometry. Analytical Biochemistry, 398, 169–177.

    Article  PubMed  CAS  Google Scholar 

  • Ershov, B. G. (1998). Radiation-chemical degradation of cellulose and other polysaccharides. Russian Chemical Reviews, 67, 315–334.

    Article  Google Scholar 

  • Faijes, M., Mars, A. E., & Smid, E. J. (2007). Comparison of quenching and extraction methodologies for metabolome analysis of Lactobacillus plantarum. Microbial Cell Factories, 6, 27–34.

    Article  PubMed  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 

  • Gonzalez, B., Francois, J., & Renaud, M. (1997). A rapid and reliable method for metabolite extraction in yeast using boiling buffered ethanol. Yeast, 13, 1347–1355.

    Article  PubMed  CAS  Google Scholar 

  • Gromova, M., & Roby, C. (2010). Toward Arabidopsis thaliana hydrophilic metabolome: Assessment of extraction methods and quantitative 1H NMR. Phys Plant, 140, 111–127.

    Article  CAS  Google Scholar 

  • Han, K. K., Richard, C., & Biserte, G. (1983). Current developments in chemical cleavage of proteins. International Journal of Biochemistry, 15, 875–884.

    Article  CAS  Google Scholar 

  • Hirai, M. Y., Sawada, Y., Kanaya, S., Kuromori, T., Kobayashi, M., Klausnitzer, R., et al. (2010). Toward genome-wide metabolotyping and elucidation of metabolic system: Metabolic profiling of large-scale bioresources. Journal of Plant Research, 123, 291–298.

    Article  PubMed  CAS  Google Scholar 

  • Kell, D. B., Brown, M., Davey, H. M., Dunn, W. B., Spasic, I., & Oliver, S. G. (2005). Metabolic footprinting and systems biology: The medium is the message. Nature Reviews in Microbiology, 3, 557–565.

    Article  CAS  Google Scholar 

  • Liu, J. Y., Li, N., Yang, J., Li, N., Qiu, H., Ai, D., et al. (2010). Metabolic profiling of murine plasma reveals an unexpected biomarker in rofecoxib-mediated cardiovascular events. Proceedings of the National Academy of Science USA, 107, 17017–17022.

    Article  CAS  Google Scholar 

  • Maharjan, R. P., & Ferenci, T. (2003). Global metabolite analysis: the influence of extraction methodology on metabolome profiles of Escherichia coli. Analytical Biochemistry, 313, 145–154.

    Article  PubMed  Google Scholar 

  • Marcus, F. (1985). Preferential cleavage at aspartyl-prolyl peptide bonds in dilute acid. International Journal of Peptide and Protein Research, 25, 542–546.

    Article  PubMed  CAS  Google Scholar 

  • Mas, S., Villas-Bôas, S. G., Hansen, M. E., Åkesson, M., & Nielsen, N. (2007). A comparison of direct infusion MS and GC–MS for metabolic footprinting of yeast mutants. Biotechnology and Bioengineering, 96, 1014–1022.

    Article  PubMed  CAS  Google Scholar 

  • Nishiumi, S., Shinohara, M., Ikeda, A., Yoshie, T., Hatano, N., Kakuyama, S., et al. (2010). Serum metabolomics as a novel diagnostic approach for pancreatic cancer. Metabolomics, 6, 518–528.

    Article  CAS  Google Scholar 

  • Oliyai, C., & Borchardt, R. T. (1993). Chemical pathways of peptide degradation.4. Pathways, kinetics, and mechanism of degradation of an aspartyl residue in a model hexapeptide. Pharmaceutical Research, 10, 95–102.

    Article  PubMed  CAS  Google Scholar 

  • Rabinowitz, J. D., & Kimball, E. (2007). Acidic acetonitrile for cellular metabolome extraction from Escherichia coli. Analytical Chemistry, 79, 6167–6173.

    Article  PubMed  CAS  Google Scholar 

  • Shin, M. H., Lee, D. Y., Liu, K. H., Fiehn, O., & Kim, K. H. (2010). Evaluation of sampling and extraction methodologies for the global metabolic profiling of Saccharophagus degradans. Analytical Chemistry, 82, 6660–6666.

    Article  PubMed  CAS  Google Scholar 

  • Smart, K. F., Aggio, R. B. M., Van Houtte, J. R., & Villas-Bôas, S. G. (2010). Analytical platform for metabolome analysis of microbial cells using methyl chloroformate derivatization followed by gas chromatography–mass spectrometry. Nature Protocols, 5, 1709–1729.

    Article  PubMed  CAS  Google Scholar 

  • van Gulik, W. M. (2010). Fast sampling for quantitative microbial metabolomics. Current Opinion in Biotechnology, 21, 27–34.

    Article  PubMed  Google Scholar 

  • Villas-Bôas, S. G. (2007). Sampling and sample preparation. In S. G. Villas-Bôas, U. Roessner, M. E. Hansen, J. Smedsgaard, & J. Nielsen (Eds.), Metabolome analysis—an introduction (pp. 39–82). New Jersey, USA: Wiley.

    Chapter  Google Scholar 

  • Villas-Bôas, S. G., & Bruheim, P. (2007). Cold glycerol–saline: The promising quenching solution for accurate intracellular metabolite analysis of microbial cells. Analytical Biochemistry, 370, 87–97.

    Article  PubMed  Google Scholar 

  • Villas-Bôas, S. G., Højer-Pedersen, J., Åkesson, M., Smedsgaard, J., & Nielsen, J. (2005a). Global metabolite analysis of yeast: Evaluation of sample preparation methods. Yeasts, 22, 1155–1169.

    Article  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 

  • Villas-Bôas, S. G., Mas, S., Åkesson, M., Smedsgaard, J., & Nielsen, J. (2005b). Mass spectrometry in metabolome analysis. Mass Spectrometry Reviews, 24, 613–646.

    Article  PubMed  Google Scholar 

  • Villas-Bôas, S. G., Moon, C. D., Noel, S., Hussein, H., Kelly, W. J., Cao, M., et al. (2008). Phenotypic characterization of transposon-inserted mutants of Clostridium proteoclasticum B316T using extracellular metabolomics. Journal of Biotechnology, 134, 55–63.

    Article  PubMed  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.

    Article  PubMed  CAS  Google Scholar 

  • Wu, T., Zivanovic, S., Hayes, D. G., & Weiss, J. (2008). Efficient reduction of chitosan molecular weight by high-intensity ultrasound: Underlying mechanism and effect of process parameters. Journal of Agriculture and Food Chemistry, 56, 5112–5119.

    Article  CAS  Google Scholar 

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Acknowledgments

We would like to thank Katya Ruggiero for fruitful data analysis discussions and the Health Research Council of New Zealand for financial support.

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Authors and Affiliations

  1. Centre for Microbial Innovation, School of Biological Sciences, The University of Auckland, 3A Symonds Street, Auckland, 1142, New Zealand

    Xavier Duportet, Raphael Bastos Mereschi Aggio, Sónia Carneiro & Silas Granato Villas-Bôas

  2. AgroParisTech, 16 rue Claude Bernard, 75005, Paris, France

    Xavier Duportet

  3. Institute for Biotechnology and Bioengineering (IBB), Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal

    Sónia Carneiro

Authors
  1. Xavier Duportet
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  2. Raphael Bastos Mereschi Aggio
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  3. Sónia Carneiro
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  4. Silas Granato Villas-Bôas
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Correspondence to Silas Granato Villas-Bôas.

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Duportet, X., Aggio, R.B.M., Carneiro, S. et al. The biological interpretation of metabolomic data can be misled by the extraction method used. Metabolomics 8, 410–421 (2012). https://doi.org/10.1007/s11306-011-0324-1

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  • DOI: https://doi.org/10.1007/s11306-011-0324-1

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