title: Time-Optimized Isotope Ratio LC-MS/MS for High-Throughput Quantification of Primary Metabolites.
abstract: 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 the12C and13C 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.
authors.affiliation: Max Planck Institute for Terrestrial Microbiology , Karl-von-Frisch-Strasse 16, 35043 Marburg, Germany.
authors.forename: Jan Christopher
authors.initials: JC
authors.lastname: Guder
authors.affiliation: Max Planck Institute for Terrestrial Microbiology , Karl-von-Frisch-Strasse 16, 35043 Marburg, Germany.
authors.forename: Thorben
authors.initials: T
authors.lastname: Schramm
authors.affiliation: Max Planck Institute for Terrestrial Microbiology , Karl-von-Frisch-Strasse 16, 35043 Marburg, Germany.
authors.forename: Timur
authors.initials: T
authors.lastname: Sander
authors.affiliation: Max Planck Institute for Terrestrial Microbiology , Karl-von-Frisch-Strasse 16, 35043 Marburg, Germany.
authors.forename: Hannes
authors.identifier: 0000-0002-6677-555X
authors.initials: H
authors.lastname: Link
mesh_terms: D002244:Carbon / Q000737:chemistry; D002247:Carbon Isotopes / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000378:metabolism; D005973:Glutamine / Q000032:analysis / Q000378:metabolism; D007553:Isotope Labeling; D053719:Tandem Mass Spectrometry
chemical_list: D002247:Carbon Isotopes; D005973:Glutamine; D002244:Carbon; C000615229:Carbon-13