title: Separation of isomeric short-chain acyl-CoAs in plant matrices using ultra-performance liquid chromatography coupled with tandem mass spectrometry.
abstract: Acyl coenzyme A (acyl-CoA) thioesters are important intermediates in cellular metabolism and being able to distinguish among them is critical to fully understanding metabolic pathways in plants. Although significant advances have been made in the identification and quantification of acyl-CoAs using liquid chromatography tandem mass spectrometry (LC-MS/MS), separation of isomeric species such as isobutyryl- and n-butyrl-CoA has remained elusive. Here we report an ultra-performance liquid chromatography (UPLC)-MS/MS method for quantifying short-chain acyl-CoAs including isomeric species n-butyryl-CoA and isobutyryl-CoA as well as n-valeryl-CoA and isovaleryl-CoA. The method was applied to the analysis of extracts of hop (Humulus lupulus) and provided strong evidence for the existence of an additional structural isomer of valeryl-CoA, 2-methylbutyryl-CoA, as well as an unexpected isomer of hexanoyl-CoA. The results showed differences in the acyl-CoA composition among varieties of Humulus lupulus, both in glandular trichomes and cone tissues. When compared with the analysis of hemp (Cannabis sativa) extracts, the contribution of isobutyryl-CoAs in hop was greater as would be expected based on the downstream polyketide products. Surprisingly, branched chain valeryl-CoAs (isovaleryl-CoA and 2-methylbutyryl-CoA) were the dominant form of valeryl-CoAs in both hop and hemp. The capability to separate these isomeric forms will help to understand biochemical pathways leading to specialized metabolites in plants.
authors.affiliation: Plant Sciences Department, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK, Canada S7N 5A8; National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK, Canada S7N 0W9. Electronic address: randy.purves@usask.ca.
authors.forename: Randy W
authors.initials: RW
authors.lastname: Purves
authors.affiliation: National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK, Canada S7N 0W9.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Ambrose
authors.affiliation: National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK, Canada S7N 0W9; Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, SK, Canada S7N 5E2.
authors.forename: Shawn M
authors.initials: SM
authors.lastname: Clark
authors.affiliation: Department of Biological Sciences, University of Manitoba, 50 Sifton Road, Winnipeg, MB, Canada R3T 2N2.
authors.forename: Jake M
authors.initials: JM
authors.lastname: Stout
authors.affiliation: National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK, Canada S7N 0W9; Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, SK, Canada S7N 5E2; Department of Botany, The University of British Columbia, #3529-6270 University Boulevard, Vancouver, BC, Canada V6T 1Z4.
authors.forename: Jonathan E
authors.initials: JE
authors.lastname: Page
mesh_terms: D000214:Acyl Coenzyme A / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000214:Acyl Coenzyme A; C050106:isobutyryl-coenzyme A; C024343:butyryl-coenzyme A; C044180:valeryl-coenzyme A; C044181:hexanoyl-coenzyme A; C017447:isovaleryl-coenzyme A
keywords: Acyl-CoAs; Hops; Metabolism; Plants; Tandem mass spectrometry; UPLC