Journal
JOURNAL OF EXPERIMENTAL BOTANY
Volume 73, Issue 9, Pages 2938-2952Publisher
OXFORD UNIV PRESS
DOI: 10.1093/jxb/erac062
Keywords
Central metabolism; ion suppression and enhancement; isomer separation; isotopic labeling; liquid chromatography-mass spectrometry; metabolite quantification; mixed-mode column chromatography; oilseeds
Categories
Funding
- National Science Foundation [IOS-1829365, DBI-1427621]
- USDA-National Institute of Food and Agriculture (USDA-NIFA) [2017-67013-26156]
- University of Illinois - Bill & Melinda Gates Foundation
- Foundation for Food & Agriculture Research
- UK government's Department for International Development [OPP1172157]
- U.S. Department of Energy (DOE)
- USDA-ARS
- DOE [DE-SC0014664]
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This study developed a sensitive liquid chromatography-mass spectrometry method that selectively enhanced the sensitivity of 27 central metabolites, especially phosphorylated sugars, in a single run. By combining hydrophilic interaction chromatography with anion exchange chromatography, the method improved the peak areas of phosphorylated sugars in plant tissues. Additionally, the method was applied to investigate metabolism in high lipid-producing tobacco leaves.
A liquid chromatography-mass spectrometry method that combines hydrophilic interaction chromatography with anion exchange chromatography leads to improved peaks for phosphorylated sugars in plant tissues. Assessing central carbon metabolism in plants can be challenging due to the dynamic range in pool sizes, with low levels of important phosphorylated sugars relative to more abundant sugars and organic acids. Here, we report a sensitive liquid chromatography-mass spectrometry method for analysing central metabolites on a hybrid column, where both anion-exchange and hydrophilic interaction chromatography (HILIC) ligands are embedded in the stationary phase. The liquid chromatography method was developed for enhanced selectivity of 27 central metabolites in a single run with sensitivity at femtomole levels observed for most phosphorylated sugars. The method resolved phosphorylated hexose, pentose, and triose isomers that are otherwise challenging. Compared with a standard HILIC approach, these metabolites had improved peak areas using our approach due to ion enhancement or low ion suppression in the biological sample matrix. The approach was applied to investigate metabolism in high lipid-producing tobacco leaves that exhibited increased levels of acetyl-CoA, a precursor for oil biosynthesis. The application of the method to isotopologue detection and quantification was considered through evaluating C-13-labeled seeds from Camelina sativa. The method provides a means to analyse intermediates more comprehensively in central metabolism of plant tissues.
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