Van Gulik Walter M, Canelas Andre B, Taymaz-Nikerel Hilal, Douma Rutger D, de Jonge Lodewijk P, Heijnen Joseph J
Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.
Methods Mol Biol. 2012;881:279-306. doi: 10.1007/978-1-61779-827-6_10.
Obtaining meaningful snapshots of the metabolome of microorganisms requires rapid sampling and immediate quenching of all metabolic activity, to prevent any changes in metabolite levels after sampling. Furthermore, a suitable extraction method is required ensuring complete extraction of metabolites from the cells and inactivation of enzymatic activity, with minimal degradation of labile compounds. Finally a sensitive, high-throughput analysis platform is needed to quantify a large number of metabolites in a small amount of sample. An issue which has often been overlooked in microbial metabolomics is the fact that many intracellular metabolites are also present in significant amounts outside the cells, and may interfere with the endometabolome measurements. Attempts to remove the extracellular metabolites with dedicated quenching methods often induce release of intracellular metabolites into the quenching solution. For eukaryotic microorganisms, leakage can be minimized by adaptation of the quenching method. For prokaryotic cells this had not yet been accomplished, so the application of a differential method whereby metabolites are measured in the culture supernatant as well as in total broth samples, to calculate the intracellular levels by subtraction, seems to be the most suitable approach. Here we present an overview of different sampling, quenching, and extraction methods developed for microbial metabolomics, described in the literature. Detailed protocols are provided for rapid sampling, quenching, and extraction for measurement of metabolites in total broth samples, washed cell samples and supernatant, to be applied for quantitative metabolomics of both eukaryotic and prokaryotic microorganisms.
要获取微生物代谢组有意义的快照，需要进行快速采样并立即淬灭所有代谢活性，以防止采样后代谢物水平发生任何变化。此外，需要一种合适的提取方法，确保从细胞中完全提取代谢物并使酶活性失活，同时使不稳定化合物的降解降至最低。最后，需要一个灵敏的高通量分析平台来定量少量样品中的大量代谢物。在微生物代谢组学中经常被忽视的一个问题是，许多细胞内代谢物在细胞外也大量存在，可能会干扰内膜代谢组的测量。试图用专门的淬灭方法去除细胞外代谢物往往会导致细胞内代谢物释放到淬灭溶液中。对于真核微生物，可以通过调整淬灭方法将泄漏降至最低。对于原核细胞，这一点尚未实现，因此应用一种差分方法，即在培养上清液和总肉汤样品中测量代谢物，通过相减来计算细胞内水平，似乎是最合适的方法。在此，我们概述了文献中为微生物代谢组学开发的不同采样、淬灭和提取方法。我们提供了详细的方案，用于快速采样、淬灭和提取，以测量总肉汤样品、洗涤后的细胞样品和上清液中的代谢物，可应用于真核和原核微生物的定量代谢组学研究。
