Parallelized small-scale production of uniformly (13)C-labeled cell extract for quantitative metabolome analysis

Analytical Biochemistry
Michael WeinerDirk Weuster-Botz

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.

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Citations

Sep 21, 2016·Philosophical Transactions. Series A, Mathematical, Physical, and Engineering Sciences·Rahul Vijay Kapoore, Seetharaman Vaidyanathan
Jan 31, 2018·Analytical Chemistry·Carlos GuijasGary Siuzdak
Jan 3, 2018·Analytical Chemistry·G A Nagana GowdaDaniel Raftery
Sep 15, 2021·Bioprocess and Biosystems Engineering·Kristin SchoppelDirk Weuster-Botz

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