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ARS Home » Midwest Area » Columbia, Missouri » Plant Genetics Research » Research » Publications at this Location » Publication #389021

Research Project: Soybean Seed Improvement Through Translational Genomics, Assessments of Elemental Carbon Metabolism, and Lipid Profiles

Location: Plant Genetics Research

Title: An efficient LC-MS method for isomer separation and detection of sugars, phosphorylated sugars, and organic acids

item KOLEY, SOMNATH - Danforth Plant Science Center
item CHU, KEVIN - Danforth Plant Science Center
item GILL, SABA - Danforth Plant Science Center
item Allen, Douglas - Doug

Submitted to: Journal of Experimental Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/15/2022
Publication Date: 2/17/2022
Citation: Koley, S., Chu, K.L., Gill, S.S., Allen, D.K. 2022. An efficient LC-MS method for isomer separation and detection of sugars, phosphorylated sugars, and organic acids. Journal of Experimental Botany. 73(9):2938–2952.

Interpretive Summary: Plants produce protein, oil, and carbohydrate in seeds that are harvested and used for food, to feed animals, and as a renewable stock to make other goods for society. The production of protein, oil, and carbohydrate in seeds is a consequence of central metabolism. When central metabolism is altered, it can lead to changes in the seed composition which increase or decrease the value of the seed. A common way to study metabolism is to examine the metabolites that are interconverted and that eventually lead to the production of protein or oil; however there are challenges with our ability to measure some of these intermediates. We developed a method that involves better separations and detection of more compounds and difficult to measure intermediates, that will be useful to better characterize metabolism so that it can be augmented for seeds that produce more protein or oil.

Technical Abstract: 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 (LC-MS) method for analyzing central metabolites on a hybrid column, where both anion-exchange and hydrophilic interaction chromatography (HILIC) ligands are embedded in the stationary phase. The LC 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 to 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 the acetyl-CoA, a precursor for oil biosynthesis. The application of the method to isotopologue detection and quantification was considered through evaluating 13C-labeled seeds from Camelina sativa. The method provides a means to analyze intermediates more comprehensively in central metabolism of plant tissues.