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Time-Optimized Isotope Ratio LC–MS/MS for High-Throughput Quantification of Primary Metabolites

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Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse 16, 35043 Marburg, Germany
Cite this: Anal. Chem. 2017, 89, 3, 1624–1631
Publication Date (Web):January 3, 2017
Copyright © 2017 American Chemical Society

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    Cellular metabolite concentrations hold information on the function and regulation of metabolic networks. However, current methods to measure metabolites are either low-throughput or not quantitative. Here we optimized conditions for liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) for quantitative measurements of primary metabolites in 2 min runs. In addition, we tested hundreds of multiple reaction monitoring (MRM) assays for isotope ratio mass spectrometry of most metabolites in amino acid, nucleotide, cofactor, and central metabolism. To systematically score the quality of LC–MS/MS data, we used the correlation between signals in the 12C and 13C channel of a metabolite. Applying two optimized LC methods to bacterial cell extracts detected more than 200 metabolites with less than 20% variation between replicates. An exhaustive spike-in experiment with 79 metabolite standards demonstrated the high selectivity of the methods and revealed a few confounding effects such as in-source fragments. Generally, the methods are suited for samples that contain metabolites at final concentrations between 1 nM and 10 μM, and they are sufficiently robust to analyze samples with a high salt content.

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b03731.

    • Algorithm for peak selection and scoring in Matlab format; Figure S1, differences between 12C/13C ratios determined with methods A and B; Figure S2, selectivity test for 79 single compounds against 180 MRMs; Figure S3, retention times in spike-in samples and filtered samples (PDF)

    • Table S1, RSDs of 40 metabolites measured with the 24 LC methods in Table 1; Table S2, 854 MRMs assays for 419 metabolites and their performance with methods A and B; Table S3, MRMs and results of 180 metabolites in the spike-in experiment; Table S4, calibration and absolute concentrations in E. coli MG1655 for 64 validated metabolites (XLSX)

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