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Title: A general method for quantification and discovery of acyl groups attached to acyl carrier proteins in fatty acid metabolism using LC-MS/MS

item NAM, JEONG-WON - Danforth Plant Science Center
item Jenkins, Lauren
item LI, JIA - Danforth Plant Science Center
item EVANS, BRADLEY - Danforth Plant Science Center
item JAWORSKI, JAN - Danforth Plant Science Center
item Allen, Douglas - Doug

Submitted to: The Plant Cell
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/27/2020
Publication Date: 4/1/2020
Citation: Nam, J., Jenkins, L.M., Li, J., Evans, B.S., Jaworski, J., Allen, D.K. 2020. A general method for quantification and discovery of acyl groups attached to acyl carrier proteins in fatty acid metabolism using LC-MS/MS. The Plant Cell. 32:820–832.

Interpretive Summary: Plant lipids are an important chemical feed stock for biofuels and are also a source of edible vegetable oils. Demand for plant oils is expected to double by 2040, therefore understanding how they are made and the biochemical controls involved in this process will be important to meeting the growing need. Lipids are mostly comprised of fatty acids that are synthesized as acyl chains attached to a protein in a process called fatty acid biosynthesis. Acyl chain lengths of 18 carbons are common to plants and the steps involved in fatty acid production are well-documented. Less is known about the regulation of this process and methods that can intricately analyze the steps directly involved are sorely needed. Since the production of fatty acids occurs by elongation of a chain while attached to a protein, we developed methods to specifically analyze the acyl-protein compositions in plant cells. This method involved cleavage of the protein to remove most of the amino acids which enabled a very direct analysis of the remaining acyl chain with sensitive quantification by liquid chromatography linked to a tandem mass spectrometer. The approach will be important for analyzing the changes in fatty acid biosynthesis that can lead to improved oil compositions for food or fuel and also judging the total production of oil within a tissue to understand the movement of carbon and resources through plant metabolism.

Technical Abstract: Acyl carrier proteins (ACPs) are the scaffolds for fatty acid biosynthesis in living systems, rendering them essential to a comprehensive understanding of lipid metabolism. However, accurate quantitative methods to assess individual acyl-ACPs do not exist. We developed a robust method to quantify acyl-ACPs to the picogram level. We successfully identified acyl-ACP elongation intermediates (3-hydroxyacyl-ACPs and 2,3-trans-enoyl-ACPs) and unexpected medium-chain (C10:1, C14:1) and polyunsaturated long-chain (C16:3) acyl-ACPs, indicating both the sensitivity of the method and how current descriptions of lipid metabolism and ACP function are incomplete. Such ACPs are likely important to medium-chain lipid production for fuels and highlight poorly understood lipid remodeling events in the chloroplast. The approach is broadly applicable to type II fatty acid synthase systems found in plants and bacteria as well as mitochondria from mammals and fungi because it capitalizes on a highly conserved Asp-Ser-Leu-Asp amino acid sequence in ACPs to which acyl groups attach. Our method allows for sensitive quantification using liquid chromatography–tandem mass spectrometry with de novo–generated standards and an isotopic dilution strategy and will fill a gap in our understanding, providing insights through quantitative exploration of fatty acid biosynthesis processes for optimal biofuels, renewable feedstocks, and medical studies in health and disease.