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Quantitative Metabolome Analysis Using Liquid Chromatography–high-resolution Mass Spectrometry by Patrick Kiefer; Jean-Charles Portais; Julia A. Vorholt is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

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In this report, we introduce a liquid chromatography single-mass spectrometry method for metabolome quantification, using the LTQ Orbitrap high-resolution mass spectrometer. Analytes were separated with hydrophilic interaction liquid chromatography. At a working resolution of 30,000 (at m/z 400), the limit of detection varied from 50 fmol to 5 pmol for 25 metabolites tested. In terms of metabolite concentration, the linearity was about 2 to 3 orders of magnitude for most compounds (R 2 > 0.99). To determine the accuracy of the system in complex sample matrices, the isotope dilution method was evaluated from mixtures of pure compounds and uniformly 13 C-labeled cell extracts. With the application of this method, quantification was possible within single runs even when the pool sizes of individual metabolites varied from 0.13 to 55.6 lM. As a case study, intracellular concentrations of central metabolites were determined for Methylobacterium extorquens AM1 during growth on two different carbon sources, methanol and succinate. Reproducible results from technical and biological repetitions were obtained that revealed significant variations of intracellular metabolite pool sizes, depend

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Author

Patrick Kiefer; Jean-Charles Portais; Julia A. Vorholt

Publisher

Elsevier Science; Elsevier ; Elsevier Inc.; Elsevier BV (ISSN 0003-2697)

Published

2008

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)