Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.
Methods Mol Biol. 2022;2349:11-39. doi: 10.1007/978-1-0716-1585-0_2.
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 quantification of the endo metabolome. Attempts to remove the extracellular metabolites with dedicated quenching methods often induce release of intracellular metabolites into the quenching solution. For eukaryotic microorganisms, this release can be minimized by adaptation of the quenching method. For prokaryotic cells, this has 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.
获得微生物代谢组的有意义的快照需要快速采样并立即停止所有代谢活动，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，同时最小化不稳定化合物的降解。最后，需要一个敏感、高通量的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，一个经常被忽视的问题是，许多细胞内代谢物也大量存在于细胞外，可能会干扰内代谢组的定量。使用专门的淬火方法去除细胞外代谢物的尝试往往会导致细胞内代谢物释放到淬火溶液中。对于真核微生物，可以通过调整淬火方法将这种释放最小化。对于原核细胞，这尚未实现，因此应用差异方法，即在培养上清液和总菌液样品中测量代谢物，通过减法计算细胞内水平，似乎是最合适的方法。本文综述了文献中报道的用于微生物代谢组学的不同采样、淬火和提取方法。提供了快速采样、淬火和提取的详细方案，用于测量总菌液样品、洗涤细胞样品和上清液中的代谢物，以应用于真核和原核微生物的定量代谢组学。
