title: Quantitative analysis of the microbial metabolome by isotope dilution mass spectrometry using uniformly 13C-labeled cell extracts as internal standards.
abstract: A novel method was developed for the quantitative analysis of the microbial metabolome using a mixture of fully uniformly (U) (13)C-labeled metabolites as internal standard (IS) in the metabolite extraction procedure the subsequent liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. This mixture of fully U (13)C-labeled metabolites was extracted from biomass of Saccharomyces cerevisiae cultivated in a fed-batch fermentation on fully U (13)C-labeled substrates. The obtained labeled cell extract contained, in principle, the whole yeast metabolome, allowing the quantification of any intracellular metabolite of interest in S. cerevisiae. We have applied the labeled cell extract as IS in the analysis of glycolytic and tricarboxylic acid (TCA) cycle intermediates in S. cerevisiae sampled in both steady-state and transient conditions following a glucose pulse. The use of labeled IS effectively reduced errors due to variations occurring in the analysis and sample processing. As a result, the linearity of calibration lines and the precision of measurements were significantly improved. Coextraction of the labeled cell extract with the samples also eliminates the need to perform elaborate recovery checks for each metabolite to be analyzed. In conclusion, the method presented leads to less workload, more robustness, and a higher precision in metabolome analysis.
authors.affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC, Delft, The Netherlands. l.wu@tnw.tudelft.nl
authors.forename: Liang
authors.initials: L
authors.lastname: Wu
authors.forename: Mlawule R
authors.initials: MR
authors.lastname: Mashego
authors.forename: Jan C
authors.initials: JC
authors.lastname: van Dam
authors.forename: Angela M
authors.initials: AM
authors.lastname: Proell
authors.forename: Jacobus L
authors.initials: JL
authors.lastname: Vinke
authors.forename: Cor
authors.initials: C
authors.lastname: Ras
authors.forename: Wouter A
authors.initials: WA
authors.lastname: van Winden
authors.forename: Walter M
authors.initials: WM
authors.lastname: van Gulik
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Heijnen
mesh_terms: D002247:Carbon Isotopes; D002457:Cell Extracts / Q000737:chemistry*; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D011865:Radioisotope Dilution Technique / Q000592:standards; D012015:Reference Standards; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods* / Q000592:standards
chemical_list: D002247:Carbon Isotopes; D002457:Cell Extracts; D005947:Glucose