Time-Optimized Isotope Ratio LC–MS/MS for High-Throughput Quantification of Primary Metabolites
| Autor: | Hannes Link, Timur Sander, Thorben Schramm, Jan Christopher Guder |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Glutamine Metabolite Tandem mass spectrometry 01 natural sciences Analytical Chemistry 03 medical and health sciences chemistry.chemical_compound Tandem Mass Spectrometry Escherichia coli Isotope-ratio mass spectrometry Chromatography High Pressure Liquid chemistry.chemical_classification Carbon Isotopes Chromatography Isotope 010401 analytical chemistry Selected reaction monitoring Primary metabolite Metabolism Carbon 0104 chemical sciences Amino acid 030104 developmental biology chemistry Isotope Labeling |
| Zdroj: | Analytical Chemistry. 89:1624-1631 |
| ISSN: | 1520-6882 0003-2700 |
| Popis: | 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 fra... |
| Databáze: | OpenAIRE |
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