title: Parallelized small-scale production of uniformly (13)C-labeled cell extract for quantitative metabolome analysis.
abstract: The need for quantitative intracellular metabolome information is central to modern applied biotechnology and systems biology. In most cases, sample preparation and metabolite analysis result in degradation of metabolites and signal suppression due to metabolite instability and matrix effects during LC-MS analysis. Therefore the application of uniformly (U) (13)C-labeled cell extract as an internal standard has gained interest in recent years. In this study a multiple-step protocol has been developed for efficient preparation of U-(13)C-labeled Escherichia coli cell extracts in stirred-tank bioreactors on a milliliter scale with a minimal supply of costly (13)C-labeled substrate. Significant reduction of fermentation medium salt concentration in the U-(13)C-labeled cell extract was achieved to reduce ion-suppression effects during mass-spectrometric analysis. Additionally, variation of reaction conditions in parallel-operated stirred-tank bioreactors on a milliliter scale enables the simultaneous preparation of U-(13)C-labeled cell extracts with varying metabolite concentrations, which is shown by an example of the labeled phosphoenolpyruvate level in E. coli.
authors.affiliation: Lehrstuhl für Bioverfahrenstechnik, Technische Universität München, 85748 Garching, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Weiner
authors.affiliation: Lehrstuhl für Bioverfahrenstechnik, Technische Universität München, 85748 Garching, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Tröndle
authors.affiliation: Lehrstuhl für Bioverfahrenstechnik, Technische Universität München, 85748 Garching, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Schmideder
authors.affiliation: Institut für Mikrobiologie, Universität Stuttgart, 70569 Stuttgart, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Albermann
authors.affiliation: Lehrstuhl für Bioverfahrenstechnik, Technische Universität München, 85748 Garching, Germany.
authors.forename: Korbinian
authors.initials: K
authors.lastname: Binder
authors.affiliation: Institut für Mikrobiologie, Universität Stuttgart, 70569 Stuttgart, Germany.
authors.forename: Georg A
authors.initials: GA
authors.lastname: Sprenger
authors.affiliation: Lehrstuhl für Bioverfahrenstechnik, Technische Universität München, 85748 Garching, Germany. Electronic address: d.weuster-botz@lrz.tum.de.
authors.forename: Dirk
authors.initials: D
authors.lastname: Weuster-Botz
mesh_terms: D019149:Bioreactors / Q000382:microbiology*; D002247:Carbon Isotopes / Q000032:analysis / Q000378:metabolism; D002457:Cell Extracts / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D005285:Fermentation; D007553:Isotope Labeling / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D055442:Metabolome; D055432:Metabolomics / Q000379:methods*
chemical_list: D002247:Carbon Isotopes; D002457:Cell Extracts
keywords: Cell extract; Isotope dilution; Mass spectrometry; Metabolomics; Parallelized milliliter bioreactors