title: Quantifying the Metabolites of the Methylerythritol 4-Phosphate (MEP) Pathway in Plants and Bacteria by Liquid Chromatography-Triple Quadrupole Mass Spectrometry.
abstract: The 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway occurs in the plastids of higher plants and in most economically important prokaryotes where it is responsible for the biosynthesis of the isoprenoid building blocks, isopentenyl diphosphate and dimethylallyl diphosphate. These five-carbon compounds are the substrates for the enormous variety of terpenoid products, including many essential metabolites and substances of commercial value. Increased knowledge of the regulation of the MEP pathway is critical to understanding many aspects of plant and microbial metabolism as well as in developing biotechnological platforms for producing these commercially valuable isoprenoids. To achieve this goal, researchers must have the ability to investigate the in vivo kinetics of the pathway by accurately measuring the concentrations of MEP pathway metabolites. However, the low levels of these metabolites complicate their accurate determination without suitable internal standards. This chapter describes a sensitive method to accurately determine the concentrations of MEP pathway metabolites occurring at trace amounts in biological samples using liquid chromatography coupled to triple quadrupole mass spectrometry. In addition, simple protocols are given for producing stable isotope-labeled internal standards for these analyses.
authors.affiliation: Max Planck Institute for Chemical Ecology, Jena, Germany.
authors.forename: D
authors.initials: D
authors.lastname: González-Cabanelas
authors.affiliation: Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa.
authors.forename: A
authors.initials: A
authors.lastname: Hammerbacher
authors.affiliation: Max Planck Institute for Chemical Ecology, Jena, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Raguschke
authors.affiliation: Max Planck Institute for Chemical Ecology, Jena, Germany. Electronic address: gershenzon@ice.mpg.de.
authors.forename: J
authors.initials: J
authors.lastname: Gershenzon
authors.affiliation: Max Planck Institute for Chemical Ecology, Jena, Germany.
authors.forename: L P
authors.initials: LP
authors.lastname: Wright
mesh_terms: D017360:Arabidopsis / Q000737:chemistry / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods*; D004896:Erythritol / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods*; D053858:Metabolic Networks and Pathways; D013403:Sugar Phosphates / Q000032:analysis / Q000378:metabolism*
chemical_list: C114232:2-C-methylerythritol 4-phosphate; D013403:Sugar Phosphates; D004896:Erythritol
keywords: 1-Deoxy-d-xylulose 5-phosphate; 2-C-methyl-d-erythitol 4-phosphate; 2-C-methyl-d-erythritol-2,4-cyclodiphosphate; Dimethylallyl diphosphate; Hydrophilic interaction liquid chromatography; Isopentenyl diphosphate; Isotope-labeled standard; Quantification; Tandem mass spectrometry