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Mashego, M. R.; Wu, L.; Van Dam, J. C.; Ras, C.; Vinke, J. L.; Van Winden, W. A.; Van Gulik, W. M.; Heijnen, J. J.

MIRACLE: mass isotopomer ratio analysis of U‐13C‐labeled extracts. A new method for accurate quantification of changes in concentrations of intracellular metabolites

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  • Medientyp: E-Artikel
  • Titel: MIRACLE: mass isotopomer ratio analysis of U‐13C‐labeled extracts. A new method for accurate quantification of changes in concentrations of intracellular metabolites
  • Beteiligte: Mashego, M. R.; Wu, L.; Van Dam, J. C.; Ras, C.; Vinke, J. L.; Van Winden, W. A.; Van Gulik, W. M.; Heijnen, J. J.
  • Erschienen: Wiley, 2004
  • Erschienen in: Biotechnology and Bioengineering, 85 (2004) 6, Seite 620-628
  • Sprache: Englisch
  • DOI: 10.1002/bit.10907
  • ISSN: 0006-3592; 1097-0290
  • Entstehung:
  • Anmerkungen:
  • Beschreibung: AbstractFirst, we report the application of stable isotope dilution theory in metabolome characterization of aerobic glucose limited chemostat culture of S. cerevisiae CEN.PK 113‐7D using liquid chromatography‐electrospray ionization MS/MS (LC‐ESI‐MS/MS). A glucose‐limited chemostat culture of S. cerevisiae was grown to steady state at a specific growth rate (μ) = 0.05 h−1 in a medium containing only naturally labeled (99% U‐12C, 1% U‐13C) carbon source. Upon reaching steady state, defined as 5 volume changes, the culture medium was switched to chemically identical medium except that the carbon source was replaced with 100% uniformly (U) 13C labeled stable carbon isotope, fed for 4 h, with sampling every hour. We observed that within a period of 1 h ∼80% of the measured glycolytic metabolites were U‐13C‐labeled. Surprisingly, during the next 3 h no significant increase of the U‐13C‐labeled metabolites occurred. Second, we demonstrate for the first time the LC‐ESI‐MS/MS‐based quantification of intracellular metabolite concentrations using U‐13C‐labeled metabolite extracts from chemostat cultivated S. cerevisiae cells, harvested after 4 h of feeding with 100% U‐13C‐labeled medium, as internal standard. This method is hereby termed “Mass Isotopomer Ratio Analysis of U‐13C Labeled Extracts” (MIRACLE). With this method each metabolite concentration is quantified relative to the concentration of its U‐13C‐labeled equivalent, thereby eliminating drawbacks of LC‐ESI‐MS/MS analysis such as nonlinear response and matrix effects and thus leads to a significant reduction of experimental error and work load (i.e., no spiking and standard additions). By coextracting a known amount of U‐13C labeled cells with the unlabeled samples, metabolite losses occurring during the sample extraction procedure are corrected for. © 2004 Wiley Periodicals, Inc.