Format

Send to

Choose Destination
J Proteome Res. 2016 May 6;15(5):1602-12. doi: 10.1021/acs.jproteome.6b00070. Epub 2016 Mar 29.

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

Author information

1
Department of Chemistry and ‡Department of Biochemistry, University of Alberta , Edmonton, Alberta, T6G 2R3 Canada.

Abstract

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 (12)C- and (13)C-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 (12)C- and (13)C-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.

KEYWORDS:

LC−MS; acid submetabolome; amine/phenol submetabolome; chemical isotope labeling; metabolomics; yeast

PMID:
26947805
DOI:
10.1021/acs.jproteome.6b00070
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for American Chemical Society
Loading ...
Support Center