LC-MS-based absolute metabolite quantification: application to metabolic flux measurement in trypanosomes.
LC-MS-based absolute metabolite quantification: application to metabolic flux measurement in trypanosomes.
复制标题
DOI：
10.1007/s11306-015-0827-2
复制
发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Barrett MP
中科院分区：
文献类型：
--
作者：
Kim DH;Achcar F;Breitling R;Burgess KE;Barrett MP
非洲人类锥虫病是一种被忽视的热带疾病，由原生动物寄生虫布氏锥虫引起。在哺乳动物的血液中，锥虫的代谢与宿主的代谢明显不同。例如，寄生虫完全依赖糖酵解作为能量来源。最近，锥虫代谢的计算和数学模型已经产生，以帮助了解寄生虫代谢，目的是促进药物开发。这些模型的优化需要定量信息，包括迄今为止无法大规模获得的代谢物浓度和/或代谢通量。在这里，我们已经实现了一种基于LC-MS的方法，该方法允许通过使用U-13 C标记的E.大肠杆菌提取物作为内标。已知量的标记E.大肠杆菌提取物加入到寄生虫样品中，以及校准标准，并用于获得校准曲线，使我们能够将强度转换为浓度。该方法使我们能够可靠地定量培养基中43种细胞内代谢物和32种细胞外代谢物随时间的变化。基于绝对量化，我们能够计算消耗和生产通量。这些定量数据现在可用于优化寄生虫代谢的计算模型。本文的在线版本（doi：10.1007/s11306-015-0827-2）包含补充材料，可供授权用户使用。
Human African trypanosomiasis is a neglected tropical disease caused by the protozoan parasite, Trypanosoma brucei. In the mammalian bloodstream, the trypanosome’s metabolism differs significantly from that of its host. For example, the parasite relies exclusively on glycolysis for energy source. Recently, computational and mathematical models of trypanosome metabolism have been generated to assist in understanding the parasite metabolism with the aim of facilitating drug development. Optimisation of these models requires quantitative information, including metabolite concentrations and/or metabolic fluxes that have been hitherto unavailable on a large scale. Here, we have implemented an LC–MS-based method that allows large scale quantification of metabolite levels by using U-13C-labelled E. coli extracts as internal standards. Known amounts of labelled E. coli extract were added into the parasite samples, as well as calibration standards, and used to obtain calibration curves enabling us to convert intensities into concentrations. This method allowed us to reliably quantify the changes of 43 intracellular metabolites and 32 extracellular metabolites in the medium over time. Based on the absolute quantification, we were able to compute consumption and production fluxes. These quantitative data can now be used to optimise computational models of parasite metabolism. The online version of this article (doi:10.1007/s11306-015-0827-2) contains supplementary material, which is available to authorized users.