MIRACLE: Mass Isotopomer Ratio Analysis of U-13C-Labeled Extracts. A New Method for Accurate Quantification of Changes in Concentrations of Intracellular Metabolites

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

*Corresponding author for this work

    Research output: Contribution to journalArticleScientificpeer-review

    228 Citations (Scopus)

    Abstract

    First, 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.

    Original languageEnglish
    Pages (from-to)620-628
    Number of pages9
    JournalBiotechnology and Bioengineering
    Volume85
    Issue number6
    DOIs
    Publication statusPublished - 2004

    Keywords

    • C labeling
    • Chemostat
    • Liquid chromatography electrospray ionization mass spectrometry
    • Quantitative metabolomics
    • Saccharomyces cerevisiae

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