Institute of Molecular Systems Biology, ETH Zurich, Switzerland.
Anal Chem. 2010 Jun 1;82(11):4403-12. doi: 10.1021/ac100101d.
Quantification of metabolites is of pivotal relevance in biology, where it complements more established omics techniques such as transcriptomics and proteomics. Here, we present a 25 min ion-pairing ultrahigh performance liquid chromatography-tandem mass spectrometry method that was developed for comprehensive coverage of central metabolism (glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle) and closely related biosynthetic reactions. We demonstrate quantification of 138 compounds, including carboxylic acids, amino acids, sugar phosphates, nucleotides, and functionalized aromatics. Biologically relevant isomers such as sugar phosphates are individually quantified by combining chromatographic separation and fragmentation. The obtained sensitivity and robustness enabled the detection of more than half all tested compounds in each of eight diverse biological samples of 0.5-50 mg dry cell weight. We recommend this method for routine and yet comprehensive quantification of primary metabolites in a wide variety of biological matrices.
代谢物的定量在生物学中具有至关重要的意义，它补充了转录组学和蛋白质组学等更成熟的组学技术。在这里，我们介绍了一种 25 分钟的离子对超高效液相色谱-串联质谱方法，该方法旨在全面涵盖中心代谢（糖酵解、戊糖磷酸途径和三羧酸循环）和密切相关的生物合成反应。我们展示了对 138 种化合物的定量分析，包括羧酸、氨基酸、糖磷酸、核苷酸和功能化芳烃。通过结合色谱分离和碎裂，对生物相关的异构体（如糖磷酸）进行单独定量。所获得的灵敏度和稳健性使我们能够在 8 种不同生物样本（干重 0.5-50mg）中的每一种中检测到超过一半的所有测试化合物。我们建议该方法用于各种生物基质中常规且全面的初级代谢物定量分析。
