Location: Plant Genetics ResearchTitle: Quantification of acyl-acyl carrier proteins for fatty acid synthesis using LC-MS/MS
|JENKINS, LAUREN - Danforth Plant Science Center|
|NAM, JEONG-WON - Chungnam National University|
|EVANS, BRADLEY - Danforth Plant Science Center|
|Allen, Douglas - Doug|
Submitted to: Methods in Molecular Biology
Publication Type: Book / Chapter
Publication Acceptance Date: 8/1/2020
Publication Date: 5/29/2021
Citation: Jenkins, L.M., Nam, J., Evans, B.S., Allen, D.K. 2021. Quantification of acyl-acyl carrier proteins for fatty acid synthesis using LC-MS/MS. Methods in Molecular Biology. 2295:219-247. https://doi.org/10.1007/978-1-0716-1362-7_13.
Interpretive Summary: Lipids are the most energy-dense major storage reserve in nature; therefore, understanding how lipids are made has important applications to human nutrition and biofuel production. The energy in lipids is a result of fatty acids that are synthesized by adding two carbons at a time to an elongating acyl chain. Though the steps are known, questions remain about the regulation and dynamic operation of this process, leaving a void in our understanding. We developed methods with isotopic labeling and mass spectrometry to rigorously quantify the involved protein and acyl chains as they were being made. As tools we developed did not previously exist, it is likely that our method will lead to many insights in the future. The work is impactful because lipids that are derived from fatty acids are important sources of fuel and serve as feed stocks for renewable needs, thus understanding better and more quantitatively the metabolic aspects of fatty acid biosynthesis will enable strategies for fuels and renewables in crops.
Technical Abstract: The fatty acid biosynthetic cycle is predicated on an acyl carrier protein (ACP) scaffold where two carbon acetyl groups are added in a chain elongation process through a series of repeated enzymatic steps. The chain extension is terminated by hydrolysis with a thioesterase or direct transfer of the acyl group to a glycerophospholipid by an acyltransferase. Methods for analysis of the concentrations of acyl chains attached to ACPs are lacking but would be informative for studies in lipid metabolism. We describe a method to profile and quantify the levels of acyl-ACPs in plants, bacteria and mitochondria of animals and fungi that represent Type II fatty acid biosynthetic systems. ACPs of Type II systems have a highly conserved Asp-Ser-Leu-Asp (DSLD) amino acid sequence at the attachment site for 4'-phosphopantetheinyl arm carrying the acyl chain. Three amino acids of the conserved sequence can be cleaved away from the remainder of the protein using an aspartyl protease. Thus, partially purified protein can be enzymatically hydrolyzed to produce an acyl chain linked to a tripeptide via the 4'-phosphopantetheinyl group. After ionization and fragmentation, the corresponding fragment ion is detected by a triple quadrupole mass spectrometer using a multiple reaction monitoring method. N isotopically labeled acyl-ACPs generated in high amounts are used with an isotope dilution strategy to quantify the absolute levels of each acyl group attached to the acyl carrier protein scaffold.