Max Planck Institute for Terrestrial Microbiology , Karl-von-Frisch-Strasse 16, 35043 Marburg, Germany.
Anal Chem. 2017 Feb 7;89(3):1624-1631. doi: 10.1021/acs.analchem.6b03731. Epub 2017 Jan 13.
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 C and 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.
细胞代谢物浓度可以提供有关代谢网络功能和调节的信息。然而，目前用于测量代谢物的方法要么通量低，要么无法定量。在这里，我们优化了液相色谱-串联质谱（LC-MS/MS）的条件，以便在 2 分钟的运行时间内对初级代谢物进行定量测量。此外，我们还测试了数百种用于氨基酸、核苷酸、辅因子和中心代谢物中大多数代谢物的同位素比质谱的多重反应监测（MRM）分析。为了系统地评分 LC-MS/MS 数据的质量，我们使用代谢物 C 通道和 C 通道信号之间的相关性。应用两种优化的 LC 方法对细菌细胞提取物进行检测，可检测到 200 多种代谢物，其重复之间的变化小于 20%。用 79 种代谢物标准品进行的全面添加实验证明了这些方法的高选择性，并揭示了一些干扰效应，如源内碎片。一般来说，这些方法适用于终浓度在 1 nM 至 10 μM 之间的样品，并且足够稳健，可以分析高盐含量的样品。
