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Metabolic Profiling of Klebsiella oxytoca: Evaluation of Methods for Extraction of Intracellular Metabolites Using UPLC/Q-TOF-MS

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Abstract

The global pool of intracellular metabolites is a reflection of all the metabolic functions of an organism. In the absence of in situ methods capable of directly measuring metabolite pools, intracellular metabolite measurements need to be performed after an extraction procedure. In this study, we evaluated the optimization of technologies for generation of a global metabolomics profile for intracellular metabolites in Klebsiella oxytoca. Intracellular metabolites of K. oxytoca were extracted at the early stationary phase using six different common extraction procedures, including cold methanol, boiling ethanol, methanol/chloroform combinations, hot water, potassium hydroxide, and perchloric acid. The metabolites were subsequently collected for further analysis, and intracellular metabolite concentration profiles were generated using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. During analysis, the stability of metabolites extracted using cold methanol was clearly higher than that obtained by other extraction methods. For the majority of metabolites, extracts generated in this manner exhibited the greatest recovery, with high reproducibility. Therefore, the use of cold ethanol was the best extraction method for attaining a metabolic profile. However, in another parallel extraction method, perchloric acid may also be required to maximize the range of metabolites recovered, particularly to extract glucose 1-phosphate and NADPH.

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References

  1. Buchholz, A., Hurlebaus, J., Wandrey, C., & Takors, R. (2002). Biomolecular Engineering, 19, 5–15.

    Article  CAS  Google Scholar 

  2. Ji, X. J., Huang, H., Li, S., Du, J., & Lian, M. (2008). Biotechnology Letters, 30, 731–734.

    Article  CAS  Google Scholar 

  3. Dunn, W. B., & Ellis, D. I. (2005). Trends in Analytical Chemistry, 24, 285–294.

    Article  CAS  Google Scholar 

  4. Dettmer, K., Aronov, P. A., & Hammock, B. D. (2007). Mass Spectrometry Reviews, 26, 51–78.

    Article  CAS  Google Scholar 

  5. Moco, S., Bino, R. J., De Vos, R. C. H., & Vervoort, J. (2007). Trends in Analytical Chemistry, 26, 855–866.

    Article  CAS  Google Scholar 

  6. Hernandez, F., Sancho, J. V., Ibanez, M., & Grimalt, S. (2008). Trends in Analytical Chemistry, 27, 862–872.

    Article  CAS  Google Scholar 

  7. Ferrer, I., & Thurman, E. M. (2003). Trends in Analytical Chemistry, 22, 750–756.

    Article  CAS  Google Scholar 

  8. Petrovic, M., Hernando, M. D., Dıaz-Cruz, M. S., & Barcelo, D. (2005). Journal of Chromatography. A, 1067, 1–14.

    Article  CAS  Google Scholar 

  9. Zhang, N., Fountain, S. T., Bi, H., & Rossi, D. T. (2000). Analytical Chemistry, 72, 800–806.

    Article  CAS  Google Scholar 

  10. Xie, G., Plumb, R., Su, M., Xu, Z., Zhao, A., Qiu, I., et al. (2008). Journal of Separation Science, 31, 1015–1026.

    Article  CAS  Google Scholar 

  11. Mihaleva, V. V., Vorst, O., Maliepaard, C., Verhoeven, H. A., de Vos, R. C. H., Hall, R. D., et al. (2008). Metabolomics, 4, 171–182.

    Article  CAS  Google Scholar 

  12. Lacorte, S., & Fernandez-Alba, A. R. (2006). Mass Spectrometry Reviews, 25, 866–880.

    Article  CAS  Google Scholar 

  13. Yao, H. B., Han, G. J., Liu, G. X., Xie, Y., & Wang, C. H. (2010). Bulletin of Environmental Contamination and Toxicology, 85, 142–146.

    Article  CAS  Google Scholar 

  14. Chen, G., Pramanik, B. N., Liu, Y., & Mirza, U. A. (2007). Journal of Mass Spectrometry, 42, 279–287.

    Article  CAS  Google Scholar 

  15. Xiao, J. F., Zhou, B., & Ressom, H. W. (2011). Trends in Analytical Chemistry, 32, 1–14.

    Article  Google Scholar 

  16. De Koning, W., & van Dam, K. (1992). Analytical Biochemistry, 204, 118–123.

    Article  Google Scholar 

  17. Lange, H. C., Eman, M., van Zuijlen, G., Visser, D., van Dam, J. C., Frank, J., et al. (2001). Biotechnology and Bioengineering, 75, 406–415.

    Article  CAS  Google Scholar 

  18. Hiller, J., Franco-Lara, E., Papaioannou, V., & Weuster-Botz, D. (2007). Biotechnology Letters, 29, 1161–1167.

    Article  CAS  Google Scholar 

  19. Smits, H. P., Cohen, A., Buttler, T., Nielsen, J., & Olsson, L. (1998). Analytical Biochemistry, 261, 36–42.

    Article  CAS  Google Scholar 

  20. Gonzalez, B., Francois, J., & Renaud, M. (1997). Yeast, 13, 1347–1356.

    Article  CAS  Google Scholar 

  21. Shryock, J. C., Rubio, R., & Berne, R. M. (1986). Analytical Biochemistry, 159, 73–81.

    Article  CAS  Google Scholar 

  22. Canelas, A. B., Pierick, A., Ras, C., Seifar, R. M., van Dam, J. C., van Gulik, W. M., et al. (2009). Analytical Chemistry, 81, 7379–7389.

    Article  CAS  Google Scholar 

  23. Maharjan, R. P., & Ferenci, T. (2003). Analytical Biochemistry, 313, 145–154.

    Article  Google Scholar 

  24. Winder, C. L., Dunn, W. B., Schuler, S., Broadhurst, D., Jarvis, R., Stephens, G. M., et al. (2008). Analytical Chemistry, 80, 2939–2948.

    Article  CAS  Google Scholar 

  25. Hans, M. A., Heinzle, E., & Wittmann, C. (2001). Applied Microbiology and Biotechnology, 56, 776–779.

    Article  CAS  Google Scholar 

  26. Ohashi, Y., Hirayama, A., Ishikawa, T., Nakamura, S., Shimizu, K., Ueno, Y., et al. (2008). Molecular BioSystems, 8, 135–147.

    Article  Google Scholar 

  27. Bennett, B., Yuan, J., Kimball, E., & Rabinowitz, J. (2008). Nature Protocols, 3, 1299–1311.

    Article  CAS  Google Scholar 

  28. Zinebi, S., Raval, G., & Petitdemange, H. (1994). Current Microbiology, 29, 79–85.

    Article  CAS  Google Scholar 

  29. Thompson, J., & Thomas, T. D. (1977). Journal of Bacteriology, 130, 583–595.

    CAS  Google Scholar 

  30. Alemohammad, M. M., & Knowles, C. J. (1974). Journal of General Microbiology, 82, 125–142.

    CAS  Google Scholar 

  31. Cook, A. M., & Fewson, C. A. (1972). Biochimica et Biophysica Acta, 290, 384–388.

    Article  CAS  Google Scholar 

  32. Harold, F. M., & Spitz, E. (1975). Journal of Bacteriology, 122, 266–277.

    CAS  Google Scholar 

  33. Thomson, J. (1976). Journal of Bacteriology, 127, 719–730.

    Google Scholar 

  34. Vaidyanathan, S., Kell, D. B., & Goodacre, R. (2002). Journal of the American Society for Mass Spectrometry, 13, 118–128.

    Article  CAS  Google Scholar 

  35. Scholz, M., Gatzek, S., Sterling, A., Fiehn, O., & Selbig, J. (2004). Bioinformatics, 20, 2447–2454.

    Article  CAS  Google Scholar 

  36. More, N., Daniel, R. M., & Petach, H. H. (1995). Biochemical Journal, 305, 17–20.

    CAS  Google Scholar 

  37. Bragger, J. M., Dunn, R. V., & Daniel, R. M. (2000). Biochimica et Biophysica Acta, 1480, 278–281.33.

    Article  CAS  Google Scholar 

  38. Villas-Boas, S. G., Hojer-Pedersen, J., Akesson, M., Smedsgaard, J., & Nielsen, J. (2005). Yeast, 22, 1155–1169.

    Article  CAS  Google Scholar 

  39. Rod, M. L., Alam, K. Y., Cunningham, P. R., & Clark, D. P. (1988). Journal of Bacteriology, 170, 3601–3610.

    CAS  Google Scholar 

  40. Lowry, O. H., Carter, J., Ward, J. B., & Glaser, L. (1971). Journal of Biological Chemistry, 246, 6511–6521.

    CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Korean Systems Biology Research Project (20110002153) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea. This research was supported by the R&D Program of MKE/KEIT (no. 10035578, Development of 2,3-butanediol and derivative production technology for C-Zero bio-platform industry).

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Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, Sogang University, Seoul, 121-742, Republic of Korea

    Changhun Park, Seokhun Yun & Jinwon Lee

  2. Research Institute of Biotechnology, CJ CheilJedang, Seoul, 157-724, Republic of Korea

    Seokhun Yun

  3. Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, Republic of Korea

    Sang Yup Lee

  4. Department of Biomolecular and Chemical Engineering, Hongik University, Chungcheongnam-do, 339-701, Republic of Korea

    Kyungmoon Park

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  1. Changhun Park
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  2. Seokhun Yun
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  4. Kyungmoon Park
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Correspondence to Jinwon Lee.

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Park, C., Yun, S., Lee, S.Y. et al. Metabolic Profiling of Klebsiella oxytoca: Evaluation of Methods for Extraction of Intracellular Metabolites Using UPLC/Q-TOF-MS. Appl Biochem Biotechnol 167, 425–438 (2012). https://doi.org/10.1007/s12010-012-9685-9

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  • DOI: https://doi.org/10.1007/s12010-012-9685-9

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