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Quantitative metabolome analysis using liquid chromatography-high-resolution mass spectrometry
Authors:Kiefer Patrick  Portais Jean-Charles  Vorholt Julia A
Institution:a Institute of Microbiology, ETH Zurich, Wolfgang-Pauli-Strasse 10, Hoenggerberg HCI F429, 8093 Zurich, Switzerland
b Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France
c INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France
d CNRS, UMR5504, F-31400 Toulouse, France
Abstract: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 (R2 > 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 13C-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 μM. 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, depending on the carbon source. The LTQ Obitrap offers new perspectives and strategies for metabolome quantification.
Keywords:Metabolome quantification  13C labeling  Isotopomer dilution method  High-resolution mass spectrometry  LC-MS  Methylobacterium
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