High-Performance Chemical Isotope Labeling Liquid Chromatography–Mass Spectrometry for Profiling the Metabolomic Reprogramming Elicited by Ammonium Limitation in Yeast

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• 作者：Xian Luo ; Shuang Zhao ; Tao Huan ; Difei Sun ; R. Magnus N. Friis ; Michael C. Schultz ; Liang Li
• 刊名：Journal of Proteome Research
• 出版年：2016
• 出版时间：May 6, 2016
• 年：2016
• 卷：15
• 期：5
• 页码：1602-1612
• 全文大小：578K
• 年卷期：0
• ISSN：1535-3907

文摘

Information about how yeast metabolism is rewired in response to internal and external cues can inform the development of metabolic engineering strategies for food, fuel, and chemical production in this organism. We report a new metabolomics workflow for the characterization of such metabolic rewiring. The workflow combines efficient cell lysis without using chemicals that may interfere with downstream sample analysis and differential chemical isotope labeling liquid chromatography mass spectrometry (CIL LC–MS) for in-depth yeast metabolome profiling. Using ¹²C- and ¹³C-dansylation (Dns) labeling to analyze the amine/phenol submetabolome, we detected and quantified a total of 5719 peak pairs or metabolites. Among them, 120 metabolites were positively identified using a library of 275 Dns-metabolite standards, and 2980 metabolites were putatively identified based on accurate mass matches to metabolome databases. We also applied ¹²C- and ¹³C-dimethylaminophenacyl (DmPA) labeling to profile the carboxylic acid submetabolome and detected over 2286 peak pairs, from which 33 metabolites were positively identified using a library of 188 DmPA-metabolite standards, and 1595 metabolites were putatively identified. Using this workflow for metabolomic profiling of cells challenged by ammonium limitation revealed unexpected links between ammonium assimilation and pantothenate accumulation that might be amenable to engineering for better acetyl-CoA production in yeast. We anticipate that efforts to improve other schemes of metabolic engineering will benefit from application of this workflow to multiple cell types.