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Evaluation of extraction processes for intracellular metabolite profiling of mammalian cells: matching extraction approaches to cell type and metabolite targets

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Abstract

In this study we report on the optimisation of the technologies for generation of a global metabolomics profile for intracellular metabolites in Chinese hamster ovary (CHO) cells. We evaluated the effectiveness of a range of different extraction methods applied to CHO cells which had been quenched using a previously optimised approach. The extraction methods tested included cold methanol, hot ethanol, acid, alkali and methanol/chloroform plus combinations of these. The extraction of metabolites using two 100% methanol extractions followed by a final water extraction recovered the largest range of metabolites. For the majority of metabolites, extracts generated in this manner exhibited the greatest recovery with high reproducibility. Therefore, this was the best extraction method for attaining a global metabolic profile from a single sample. However, another parallel extraction method (e.g. alkali) may also be required to maximise the range of metabolites recovered (e.g. non-polar metabolites).

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Abbreviations

CHO:

Chinese hamster ovary

AMBIC:

Ammonium bicarbonate

PCA:

Perchloric acid

GC-MS:

Gas chromatography-mass spectrometry

References

  • Allen, J., Davey, H. M., Broadhurst, D., Heald, J. K., Rowland, J. J., Oliver, S. G., et al. (2003). High-throughput classification of yeast mutants for functional genomics using metabolic footprinting. Nature Biotechnology, 21, 692–696.

    Article  CAS  PubMed  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  CAS  PubMed  Google Scholar 

  • Biais, B., Allwood, J. W., Deborde, C., Xu, Y., Maucourt, M., Beauvoit, B., et al. (2009). 1H NMR, GC-EI-TOFMS, and data set correlation for fruit metabolomics: Application to spatial metabolite analysis in melon. Analytical Chemistry, 81, 2884–2894.

    Article  CAS  PubMed  Google Scholar 

  • Birch, J. R., & Racher, A. J. (2006). Antibody production. Advanced Drug Delivery Reviews, 58, 671–685.

    Article  CAS  PubMed  Google Scholar 

  • Bundy, J. G., Papp, B., Harmston, R., Browne, R. A., Clayson, E. M., Burton, N., et al. (2007). Evaluation of predicted network modules in yeast metabolism using NMR-based metabolite profiling. Genome Research, 17, 510–519.

    Article  CAS  PubMed  Google Scholar 

  • Butler, M. (2005). Animal cell cultures: Recent achievements and perspectives in the production of biopharmaceuticals. Applied Microbiology and Biotechnology, 68, 283–291.

    Article  CAS  PubMed  Google Scholar 

  • Chassagnole, C., Noisommit-Rizzi, N., Schmid, J. W., Mauch, K., & Reuss, M. (2002). Dynamic modeling of the central carbon metabolism of Escherichia coli. Biotechnology and Bioengineering, 79, 53–73.

    Article  CAS  PubMed  Google Scholar 

  • Entian, K. D., Zimmermann, F. K., & Scheel, I. (1977). A partial defect in carbon catabolite repression in mutants of Saccharomyces cerevisiae with reduced hexose phosphorylation. Molecular and General Genetics, 156, 99–105.

    Article  CAS  PubMed  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.

    Article  PubMed  Google Scholar 

  • Fiehn, O. (2002). Metabolomics—The link between genotypes and phenotypes. Plant Molecular Biology, 48, 155–171.

    Article  CAS  PubMed  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  CAS  PubMed  Google Scholar 

  • Goodacre, R. (2007). Metabolomics of a superorganism. Journal of Nutrition, 137, 259S–266S.

    CAS  PubMed  Google Scholar 

  • Hajjaj, H., Blanc, P. J., Goma, G., & Francois, J. (1998). Sampling techniques and comparative extraction procedures for quantitative determination of intra- and extracellular metabolites in filamentous fungi. FEMS Microbiology Letters, 164, 195–200.

    Article  CAS  Google Scholar 

  • Heazell, A. E., Brown, M., Dunn, W. B., Worton, S. A., Crocker, I. P., Baker, P. N., et al. (2008). Analysis of the metabolic footprint and tissue metabolome of placental villous explants cultured at different oxygen tensions reveals novel redox biomarkers. Placenta, 29, 691–698.

    Article  CAS  PubMed  Google Scholar 

  • Hofmann, U., Maier, K., Niebel, A., Vacun, G., Reuss, M., & Mauch, K. (2008). Identification of metabolic fluxes in hepatic cells from transient 13C-labeling experiments: Part I. Experimental observations. Biotechnology and Bioengineering, 100, 344–354.

    Article  CAS  PubMed  Google Scholar 

  • Hollywood, K., Brison, D. R., & Goodacre, R. (2006). Metabolomics: Current technologies and future trends. Proteomics, 6, 4716–4723.

    Article  CAS  PubMed  Google Scholar 

  • Kenny, L. C., Broadhurst, D., Brown, M., Dunn, W. B., Redman, C. W., Kell, D. B., et al. (2008). Detection and identification of novel metabolomic biomarkers in preeclampsia. Reproductive Sciences, 15, 591–597.

    Article  CAS  PubMed  Google Scholar 

  • Khoo, S. H., & Al-Rubeai, M. (2009). Metabolic characterization of a hyper-productive state in an antibody producing NS0 myeloma cell line. Metabolic Engineering, 11, 199–211.

    Article  CAS  PubMed  Google Scholar 

  • Ma, N., Ellet, J., Okediadi, C., Hermes, P., Mccormick, E., & Casnocha, S. (2009). A single nutrient feed supports both chemically defined NS0 and CHO fed-batch processes: Improved productivity and lactate metabolism. Biotechnology Progress, 25(5), 1353–1363.

    Article  CAS  PubMed  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 

  • Mounet, F., Lemaire-Chamley, M., Maucourt, M., Cabasson, C., Giraudel, J. L., Deborde, C., et al. (2007). Quantitative metabolic profiles of tomato flesh and seeds during fruit development: Complementary analysis with ANN and PCA. Metabolomics, 3, 273–288.

    Article  CAS  Google Scholar 

  • Park, S. J., Lee, S. Y., Cho, J., Kim, T. Y., Lee, J. W., Park, J. H., et al. (2005). Global physiological understanding and metabolic engineering of microorganisms based on omics studies. Applied Microbiology and Biotechnology, 68, 567–579.

    Article  CAS  PubMed  Google Scholar 

  • Ritter, J. B., Genzel, Y., & Reichl, U. (2008). Simultaneous extraction of several metabolites of energy metabolism and related substances in mammalian cells: Optimization using experimental design. Analytical Biochemistry, 373, 349–369.

    Article  CAS  PubMed  Google Scholar 

  • Sellick, C. A., Hansen, R., Maqsood, A. R., Dunn, W. B., Stephens, G. M., Goodacre, R., et al. (2009). Effective quenching processes for physiologically valid metabolite profiling of suspension cultured Mammalian cells. Analytical Chemistry, 81, 174–183.

    Article  CAS  PubMed  Google Scholar 

  • Sreekumar, A., Poisson, L. M., Rajendiran, T. M., Khan, A. P., Cao, Q., Yu, J. D., et al. (2009). Metabolomic profiles delineate potential role for sarcosine in prostate cancer progression. Nature, 457, 910–914.

    Article  CAS  PubMed  Google Scholar 

  • Sumner, L. W., Amberg, A., Barrett, D., Beale, M. H., Beger, R., Daykin, C. A., et al. (2007). Proposed minimum reporting standards for chemical analysis. Metabolomics, 3, 211–221.

    Article  CAS  Google Scholar 

  • Teng, Q., Huang, W. L., Collette, T. W., Ekman, D. R., & Tan, C. (2009). A direct cell quenching method for cell-culture based metabolomics. Metabolomics, 5, 199–208.

    Article  CAS  Google Scholar 

  • Theobald, U., Mailinger, W., Reuss, M., & Rizzi, M. (1993). In vivo analysis of glucose-induced fast changes in yeast adenine nucleotide pool applying a rapid sampling technique. Analytical Biochemistry, 214, 31–37.

    Article  CAS  PubMed  Google Scholar 

  • Tweeddale, H., Notley-Mcrobb, L., & Ferenci, T. (1998). Effect of slow growth on metabolism of Escherichia coli, as revealed by global metabolite pool (“metabolome”) analysis. Journal of Bacteriology, 180, 5109–5116.

    CAS  PubMed  Google Scholar 

  • Tweeddale, H., Notley-Mcrobb, L., & Ferenci, T. (1999). Assessing the effect of reactive oxygen species on Escherichia coli using a metabolome approach. Redox Report, 4, 237–241.

    Article  CAS  PubMed  Google Scholar 

  • Weibel, K. E., Mor, J. R., & Fiechter, A. (1974). Rapid sampling of yeast cells and automated assays of adenylate, citrate, pyruvate and glucose-6-phosphate pools. Analytical Biochemistry, 58, 208–216.

    Article  CAS  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  CAS  PubMed  Google Scholar 

  • Wu, H., Southam, A. D., Hines, A., & Viant, M. R. (2008). High-throughput tissue extraction protocol for NMR- and MS-based metabolomics. Analytical Biochemistry, 372, 204–212.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We gratefully acknowledge the financial support from the UK BBSRC, EPSRC and industrial members of the Bioprocessing Research Industry Club (BRIC) and from a BBSRC Research Development Fellowship to GMS. We would like to thank William Allwood for helpful discussions.

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Authors and Affiliations

  1. Faculty of Life Sciences, The University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK

    Christopher A. Sellick, David Knight, Alexandra S. Croxford, Arfa R. Maqsood & Alan J. Dickson

  2. The School of Chemical Engineering and Analytical Sciences, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK

    Gill M. Stephens

  3. The School of Chemistry, Manchester Interdisciplinary Biocentre, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK

    Royston Goodacre

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  1. Christopher A. Sellick
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  2. David Knight
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  3. Alexandra S. Croxford
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  4. Arfa R. Maqsood
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  5. Gill M. Stephens
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  6. Royston Goodacre
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  7. Alan J. Dickson
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Correspondence to Christopher A. Sellick.

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Sellick, C.A., Knight, D., Croxford, A.S. et al. Evaluation of extraction processes for intracellular metabolite profiling of mammalian cells: matching extraction approaches to cell type and metabolite targets. Metabolomics 6, 427–438 (2010). https://doi.org/10.1007/s11306-010-0216-9

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  • DOI: https://doi.org/10.1007/s11306-010-0216-9

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