Mülleder Michael, Bluemlein Katharina, Ralser Markus
Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, Cambridge CB2 1GA, United Kingdom.
Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, Cambridge CB2 1GA, United Kingdom;
Cold Spring Harb Protoc. 2017 Sep 1;2017(9):pdb.prot089094. doi: 10.1101/pdb.prot089094.
Amino acids are the building blocks for protein synthesis and the precursors for many biomolecules, such as glutathione and -adenosylmethionine. Their intracellular concentrations provide valuable information about the overall metabolic state of the cell, as they are closely connected to carbon and nitrogen metabolism and are tightly regulated to meet cellular demands in ever-changing environments. Here, we describe a fast and simple method enabling metabolic profiling for free amino acids for large numbers of yeast strains. Metabolites are extracted with boiling ethanol and, without further conditioning, analyzed by hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS). Several hundred samples can be prepared in a single day with an analytical runtime of 3.25 min. This method is valuable for functional characterization, identification of metabolic regulators and processes, or monitoring of biotechnological processes.
氨基酸是蛋白质合成的基本组成单位，也是许多生物分子（如谷胱甘肽和S-腺苷甲硫氨酸）的前体。它们在细胞内的浓度提供了有关细胞整体代谢状态的有价值信息，因为它们与碳和氮代谢密切相关，并受到严格调控以满足不断变化的环境中的细胞需求。在这里，我们描述了一种快速简单的方法，可对大量酵母菌株的游离氨基酸进行代谢谱分析。代谢物用沸腾乙醇提取，无需进一步处理，通过亲水相互作用色谱-串联质谱（HILIC-MS/MS）进行分析。一天内可以制备数百个样品，分析运行时间为3.25分钟。该方法对于功能表征、代谢调节剂和过程的鉴定或生物技术过程的监测具有重要价值。
