1 research outputs found
Time-Optimized Isotope Ratio LC–MS/MS for High-Throughput Quantification of Primary Metabolites
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 <sup>12</sup>C and <sup>13</sup>C 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