Mass isotopomer ratio analysis of U- super(13)C-labeled extracts. A new method for accurate quantification of changes in concentrations of intracellular metabolites

• Published in: Biotechnology and bioengineering 2004-01, Vol.85 (6), p.620-628
• Main Authors: Mashego, M R, Wu, L, Van Dam, JC, Ras, C, Vinke, J L, Van Winden, WA, Van Gulik, WM, Heijnen, J J
• Format: Article
• Language: English
• Subjects: Saccharomyces cerevisiae
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.10907

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 ( mu ) = 0.05 h super(-1) in a medium containing only naturally labeled (99% U- super(12)C, 1% U- super(13)C) 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) super(13)C labeled stable carbon isotope, fed for 4 h, with sampling every hour. We observed that within a period of 1 h similar to 80% of the measured glycolytic metabolites were U- super(13)C-labeled. Surprisingly, during the next 3 h no significant increase of the U- super(13)C- labeled metabolites occurred. Second, we demonstrate for the first time the LC- ESI-MS/MS-based quantification of intracellular metabolite concentrations using U- super(13)C-labeled metabolite extracts from chemostat cultivated S. cerevisiae cells, harvested after 4 h of feeding with 100% U- super(13)C-labeled medium, as internal standard. This method is hereby termed "Mass Isotopomer Ratio Analysis of U- super(13)C Labeled Extracts" (MIRACLE). With this method each metabolite concentration is quantified relative to the concentration of its U- super(13)C-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- super(13)C labeled cells with the unlabeled samples, metabolite losses occurring during the sample extraction procedure are corrected for.