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

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 (mu)=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 approximately 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.

Global metabolite analysis of yeast: evaluation of sample preparation methods

“…Similar conclusions were also achieved for filamentous fungi (Hajjaj et al, 1998) and bacterial cells (Maharjan and Ferenci, 2003). The recent methodologies developed to acquire accurate information on microbial metabolite levels (Buchholz et al, 2002;Soga et al, 2002;van Dam et al, 2002;Mashego et al, 2004;Villas-Bôas et al, 2005; and others) urgently need an extraction protocol that is able to trap a higher diversity of intracellular metabolites of yeasts. However, the yeast metabolome comprises a variety of compounds with a wide range of physical and chemical properties that make this task virtually impossible.…”

“…Therefore, it is important to determine the intracellular levels of metabolites accurately, which requires efficient and reliable methods for sample preparation. There have been considerable efforts to develop sensitive and accurate methods for detection of metabolites (Buchholz et al, 2002;Soga et al, 2002;van Dam et al, 2002;Mashego et al, 2004;Villas-Bôas et al, 2005;and others) and to improve the efficiency of data mining and data analysis (Goodacre et al, 2004;Kell, 2004;van der Greef et al, 2004) but relative little attention has been given to sample preparation procedures. Figure 1 summarizes the main steps in sample preparation for metabolome analysis.…”

Development and application of a differential method for reliable metabolome analysis in Escherichia coli

“…In vivo kinetic studies rely partly on rapid dynamic perturbation experiments during which a steady-state chemostat culture is perturbed and the subsequent response in both extracellular and intracellular metabolites is monitored (Theobald et al, 1997;Chassagnole et al, 2002;Visser et al, 2002;Mashego et al, 2004;Wu et al, 2005). Taking into account the high turnover rate of most intracellular metabolites, proper measurement of their concentrations requires rapid broth sampling and instantaneous quenching of enzyme activity.…”

“…For application of LC-MS/MS especially the rigorous use of stable isotope (e.g. 13 C) labeled internal standards is a prerequisite to obtain reproducible and reliable results (Mashego et al, 2004;Wu et al, 2005). The aim of the present study was to obtain a fast and reliable method for sampling and quenching of E. coli K12 cells, which would also be applicable to highly dynamic pulse response studies, carried out in chemostat cultures.…”

Conservation of the metabolomic response to starvation across two divergent microbes

“…The concentrations and fluxes of metabolites depend on the interactions among this conserved network structure, the cellular environment, and species-specific factors, such as the location, activities, and regulation of metabolic enzymes. Except for a few classic examples, e.g., central carbon metabolism in E. coli and yeast (3)(4)(5) and nitrogen assimilation in bacteria (6-8) the effect of environmental nutrient perturbations on the cellular metabolome have not been directly measured.…”