Advertisement

Metabolomics

, Volume 11, Issue 2, pp 286–301 | Cite as

Fast sampling for quantitative microbial metabolomics: new aspects on cold methanol quenching: metabolite co-precipitation

  • Maksim Zakhartsev
  • Oliver Vielhauer
  • Thomas Horn
  • Xuelian Yang
  • Matthias Reuss
Original Article

Abstract

The intra- and extracellular concentrations of 16 metabolites were measured in chemostat (D = 0.1 h−1) anaerobic cultures of the yeast Saccharomyces cerevisiae CEN.PK-113-7D growing on minimal medium. Two independent sampling workflows were employed: (i) conventional cold methanol quenching and (ii) a differential approach. Metabolites were quantified in different sample fractions (total, extracellular, quenching supernatant, methanol/water extract and pellet) in order to derive their mass balance. The differential method in combination with absolute metabolite quantification by gas-chromatography with isotope dilution mass spectrometry (GC–IDMS) was used as a benchmark to assess quality of the cold methanol quenching procedure. Quantitative comparison of metabolite concentrations in all fractions collected by different quenching techniques indicates asystematic loss of the total mass of various metabolites in course of the cold methanol quenching. Pellet resulting from the cold methanol quenching besides biomass contains considerable amounts of precipitated inorganic salts from the fermentation media. Quantitative analysis has revealed significant co-precipitation of polar extracellular metabolites together with these salts. This phenomenon is especially significant for metabolites with large extracellular mass-fraction. We report that the co-precipitation is a hitherto neglected phenomenon and concluded that its degree strongly linked to culturing conditions (i.e. media composition) and chemical properties of the particular metabolite. Thus, intracellular metabolite levels measured from samples collected by cold methanol quenching might be uncertain and variably biased due to corruption by described phenomena.

Keywords

Yeast Sampling techniques Quantitative metabolite measurements 

Abbreviations

PCA

Perchloric acid

MES

2-(N-morpholino)ethanesulfonic acid

3PG

3-Phosphoglycerate [ChEBI:17794]

ADP

Adenosine diphosphate [ChEBI:16761]

aKG

2-Oxoglutarate [ChEBI:16810]

AMP

Adenosine monophosphate [ChEBI:16027]

ATP

Adenosine triphosphate [ChEBI:15422]

Cit

Citric acid [ChEBI:30769]

EtOH

Ethanol [ChEBI:16236]

F6P

Fructose-6-phosphate [ChEBI:16084]

Fum

Fumaric acid [ChEBI:18012]

G3P

Glycerone phosphate [ChEBI:16108]

G6P

Glucose-6-phosphate [ChEBI:17665]

GC-IDMS

Gas-chromatography with isotope dilution mass spectrometry

GTP

Guanosine triphosphate [ChEBI:15996]

gDW

Gram of dry weight biomass

IS

Internal standard

Mal

Malic acid [ChEBI:30797]

MeOH

Methanol

PEP

Phosphoenolpyruvate [ChEBI:18021]

R5P

Ribose-5-phosphate [ChEBI:17797]

Suc

Succinic acid [ChEBI:15741]

UTP

Uridine triphosphate [ChEBI:15713]

T

Total content of metabolites in both extra- and intracellular fractions

EX

Extracellular fraction of metabolites

IN

Intracellular fraction of metabolites

QS

Quenching supernatant from cold methanol method

ME

Methanol extract from cold methanol method

P

Pellet from cold methanol method

Notes

Acknowledgments

The authors would like to thank Lara Bogner, Andreas Freund, Achim Hauck, Mira Lenfers-Lücker and Alexander Müller (Institute of Biochemical Engineering (IBVT), University of Stuttgart, Germany) for the experimental support. The research was funded by the transnational research initiative “Systems Biology of Microorganisms (SysMO)” within network MOSES: “MicroOrganism Systems Biology: Energy and Saccharomyces cerevisiae” [http://www.sysmo.net/].

Supplementary material

11306_2014_700_MOESM1_ESM.docx (14 kb)
Supplementary material 1 (DOCX 13 kb)

References

  1. Bhattacharya, M., Fuhrman, L., Ingram, A., Nickerson, K. W., & Conway, T. (1995). Single-run separation and detection of multiple metabolic intermediates by anion-exchange high-performance liquid-chromatography and application to cell pool extracts prepared from Escherichia coli. Analytical Biochemistry, 232(1), 98–106.CrossRefPubMedGoogle Scholar
  2. Bolten, C. J., Kiefer, P., Letisse, F., Portais, J. C., & Wittmann, C. (2007). Sampling for metabolome analysis of microorganisms. Analytical Chemistry, 79(10), 3843–3849.CrossRefPubMedGoogle Scholar
  3. Bolten, C., & Wittmann, C. (2008). Appropriate sampling for intracellular amino acid analysis in five phylogenetically different yeasts. Biotechnology Letters, 30(11), 1993–2000.CrossRefPubMedGoogle Scholar
  4. Canelas, A., Ras, C., ten Pierick, A., et al. (2008). Leakage-free rapid quenching technique for yeast metabolomics. Metabolomics, 4(3), 226–239.CrossRefGoogle Scholar
  5. Canelas, A. Á. B., ten Pierick, A., Ras, C., et al. (2009). Quantitative evaluation of intracellular metabolite extraction techniques for yeast metabolomics. Analytical Chemistry, 81(17), 7379–7389.CrossRefPubMedGoogle Scholar
  6. de Koning, W., & van Dam, K. (1992). A method for the determination of changes of glycolytic metabolites in yeast on a subsecond time scale using extraction at neutral pH. Analytical Biochemistry, 204(1), 118–123.CrossRefPubMedGoogle Scholar
  7. Gonzalez, B., Francois, J., & Renaud, M. (1997). A rapid and reliable method for metabolite extraction in yeast using boiling buffered ethanol. Yeast, 13(14), 1347–1355.CrossRefPubMedGoogle Scholar
  8. 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(1), 195–200.CrossRefGoogle Scholar
  9. Hiller, J., Franco-Laro, E., & Weuster-Botz, D. (2007). Metabolic profiling of Escherichia coli cultivations: evaluation of extraction and metabolite analysis procedure. Biotechnology Letters, 29(8), 1169–1178.CrossRefPubMedGoogle Scholar
  10. Hofmann, U., Maier, K., Niebel, A., et al. (2008). Identification of metabolic fluxes in hepatic cells from transient 13C-labeling experiments: Part I Experimental observations. Biotechnology and Bioengineering, 100(2), 344–354.CrossRefPubMedGoogle Scholar
  11. Jensen, N. B., Jokumsen, K. V., & Villadsen, J. (1999). Determination of the phosphorylated sugars of the Embden–Meyerhoff–Parnas pathway in Lactococcus lactis using a fast sampling technique and solid phase extraction. Biotechnology and Bioengineering, 63, 357–362.CrossRefGoogle Scholar
  12. Lange, H. C., Eman, M., van Zuijlen, G., et al. (2001). Improved rapid sampling for in vivo kinetics of intracellular metabolites in Saccharomyces cerevisiae. Biotechnology and Bioengineering, 75(4), 406–415.CrossRefPubMedGoogle Scholar
  13. Letisse, F., & Lindley, N. D. (2000). An intracellular metabolite quantification technique applicable to polysaccharide-producing bacteria. Biotechnology Letters, 22(21), 1673–1677.CrossRefGoogle Scholar
  14. Loret, M. O., Pedersen, L., & Francois, J. (2007). Revised procedures for yeast metabolites extraction: application to a glucose pulse to carbon-limited yeast cultures, which reveals a transient activation of the purine salvage pathway. Yeast, 24(1), 47–60.CrossRefPubMedGoogle Scholar
  15. Maharjan, P. R., & Ferenci, T. (2003). Global metabolite analysis: the influence of extraction methodology on metabolome profiles of Escherichia coli. Analytical Biochemistry, 313(1), 145–154.CrossRefPubMedGoogle Scholar
  16. Mashego, M. R., Jansen, M. L. A., Vinke, J. L., Van Gulik, W. M., & Heijnen, J. J. (2005). Changes in the metabolome of Saccharomyces cerevisiae associated with evolution in aerobic glucose-limited chemostats. FEMS Yeast Research, 5(4–5), 419–430.CrossRefPubMedGoogle Scholar
  17. Mashego, M. R., Rumbold, K., de Mey, M., et al. (2007). Microbial metabolomics: past, present and future methodologies. Biotechnology Letters, 29(1), 1–16.CrossRefPubMedGoogle Scholar
  18. Mashego, M. R., Van Gulik, W. M., Vinke, J. L., & Heijnen, J. J. (2003). Critical evaluation of sampling techniques for residual glucose determination in carbon-limited chemostat culture of Saccharomyces cerevisiae. Biotechnology and Bioengineering, 83(4), 395–399.CrossRefPubMedGoogle Scholar
  19. Mashego, M. R., van Gulik, W. M., Vinke, J. L., Visser, D., & Heijnen, J. J. (2006). In vivo kinetics with rapid perturbation experiments in Saccharomyces cerevisiae using a second-generation BioScope. Metabolic Engineering, 8(4), 370–383.CrossRefPubMedGoogle Scholar
  20. Mashego, M. R., Wu, L., Van Dam, J. C., et al. (2004). MIRACLE: mass isotopomer ratio analysis of U-13C-labeled extracts. A new method for accurate quantification of changes in concentrations of intracellular metabolites. Biotechnology and Bioengineering, 85(6), 620–628.CrossRefPubMedGoogle Scholar
  21. Navon, G., Shulman, R. G., Yamane, T., et al. (1979). Phosphorus-31 nuclear magnetic resonance studies of wild-type and glycolytic pathway mutants of Saccharomyces cerevisiae. Biochemistry, 18(21), 4487–4499.CrossRefPubMedGoogle Scholar
  22. Reuss, M. (1991). Structured Modeling of Bioreactors. Annals of the New York Academy of Sciences, 646, 284–299.CrossRefGoogle Scholar
  23. Schaub, J., & Reuss, M. (2008). In vivo dynamics of glycolysis in Escherichia coli shows need for growth-rate dependent metabolome analysis. Biotechnology Progress, 24(6), 1402–1407.CrossRefPubMedGoogle Scholar
  24. Schaub, J., Schiesling, C., Reuss, M., & Dauner, M. (2006). Integrated sampling procedure for metabolome analysis. Biotechnology Progress, 22(5), 1434–1442.CrossRefPubMedGoogle Scholar
  25. Schellinger, A. P., & Carr, P. W. (2004). Solubility of buffers in aqueous-organic eluents for reversedphased liquid chromatography. LCGC North America, 22(6), 545–548.Google Scholar
  26. Sumner, L., et al. (2007). Proposed minimum reporting standards for chemical analysis. Metabolomics, 3, 211–221.CrossRefPubMedCentralPubMedGoogle Scholar
  27. Taymaz-Nikerel, H., de Mey, M., Ras, C., et al. (2009). Development and application of a differential method for reliable metabolome analysis in Escherichia coli. Analytical Biochemistry, 386(1), 9–19.CrossRefPubMedGoogle Scholar
  28. Theobald, U., Mailinger, W., Baltes, M., Rizzi, M., & Reuss, M. (1997). In vivo analysis of metabolic dynamics in Saccharomyces cerevisiae : I. Experimental observations. Biotechnology and Bioengineering, 55(2), 305–316.CrossRefPubMedGoogle Scholar
  29. 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(1), 31–37.CrossRefPubMedGoogle Scholar
  30. Toennessen, S. (2008). Etablierung einer GC/MS Methode zur Quantifizierung intrazellularer Metabolite in E. coli im Fed-Batch Prozess. Thesis/Dissertation. Fakultat Angewandte Naturwissenschaften, Hochschule Esslingen. Esslingen.Google Scholar
  31. van der Werf, M. J., Takors, R., Smedsgaard, J., et al. (2007). Standard reporting requirements for biological samples in metabolomics experiments: microbial and in vitro biology experiments. Metabolomics, 3(3), 189–194.CrossRefPubMedCentralPubMedGoogle Scholar
  32. van Gulik, W. M., Canelas, A. B., Seifar, R. M., & Heijnen, J. J. (2013). The sampling and sample preparation problem in microbial metabolomics metabolomics in practice (pp. 1–19). Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA.Google Scholar
  33. Vaseghi, S., Baumeister, A., Rizzi, M., & Reuss, M. (1999). In vivo dynamics of the pentose phosphate pathway in Saccharomyces cerevisiae. Metabolic Engineering, 1(2), 128–140.CrossRefPubMedGoogle Scholar
  34. Verduyn, C., Postma, E., Scheffers, W. A., & van Dijken, J. P. (1992). Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast, 8(7), 501–517.CrossRefPubMedGoogle Scholar
  35. Vielhauer, O., Zakhartsev, M., Horn, T., Takors, R., & Reuss, M. (2011). Simplified absolute metabolite quantification by GC-IDMS on the basis of commercially available source material. Journal of Chromatography B Biomedical Applications, 879(32), 3859–3870.CrossRefGoogle Scholar
  36. Villas-Boas, S. G., Hojer-Pedersen, J., Akesson, M., Smedsgaard, J., & Nielsen, J. (2005). Global metabolite analysis of yeast: evaluation of sample preparation methods. Yeast, 22(14), 1155–1169.CrossRefPubMedGoogle Scholar
  37. Visser, D., van Zuylen, G. A., Van Dam, J. C., et al. (2004). Analysis of in vivo kinetics of glycolysis in aerobic Saccharomyces cerevisiae by application of glucose and ethanol pulses. Biotechnology and Bioengineering, 88(2), 157–167.CrossRefPubMedGoogle Scholar
  38. Wittmann, C., Kromer, J. O., Kiefer, P., Binz, T., & Heinzle, E. (2004). Impact of the cold shock phenomenon on quantification of intracellular metabolites in bacteria. Analytical Biochemistry, 327(1), 135–139.CrossRefPubMedGoogle Scholar
  39. Wu, L., Mashego, M. R., Van Dam, J. C., et al. (2005). Quantitative analysis of the microbial metabolome by isotope dilution mass spectrometry using uniformly C-13-labeled cell extracts as internal standards. Analytical Biochemistry, 336(2), 164–171.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Maksim Zakhartsev
    • 1
    • 3
    • 4
  • Oliver Vielhauer
    • 1
  • Thomas Horn
    • 1
  • Xuelian Yang
    • 2
    • 1
  • Matthias Reuss
    • 1
    • 3
  1. 1.Institute of Biochemical EngineeringUniversity of StuttgartStuttgartGermany
  2. 2.Beijing Engineering and Technology Research Center of Food AdditivesBeijing Technology & Business University (BTBU)BeijingChina
  3. 3.Center Systems BiologyUniversity of StuttgartStuttgartGermany
  4. 4.Plant Systems BiologyUniversity of HohenheimStuttgartGermany

Personalised recommendations