title: Optimization of a gas chromatography-mass spectrometry method with methyl chloroformate derivatization for quantification of amino acids in plant tissue.
abstract: Rapid, easy and reliable quantification of amino acids is crucial in research on plant amino acid metabolism and nutritional improvement of crops via enrichment of essential amino acids. A recently reported analysis method, based on solid phase extraction (SPE), derivatization with methyl chloroformate and gas chromatography-mass spectrometry was optimized and tested on three-week-old Arabidopsis thaliana leaf tissues. Optimization of the SPE cleanup yielded recovery rates of minimum 95% for all amino acids (except arginine). Variations in accuracy and precision did not exceed 12.5%, except for cysteine, histidine and tryptophane, which were excluded from analysis. Quantification of overlapping peaks for isoleucine/threonine and proline/asparagine was possible by selection of two specific fragment ions for each amino acid. Of the 16 selected amino acids, 14 were quantified successfully in at least 75% of the samples, while methionine and tyrosine were only quantifiable in 6% and 42%, respectively. A case study on the aspartate super pathway confirmed the applicability of the optimized method on wild type and genetically modified plants: external supplementation of methionine or lysine yielded a 146-fold or 27-fold increase in the respective absolute amino acid levels compared with the control treatment. Induced expression of dhdps-r1 (a mutated lysine biosynthesis gene encoding a feedback insensitive enzyme) caused an 83-fold increase in absolute lysine levels.
authors.affiliation: Laboratory of Plant Genetics (PLAN), Vrije Universiteit Brussel, Brussels, Belgium.
authors.forename: Bram
authors.initials: B
authors.lastname: Vancompernolle
authors.affiliation: Department of Analytical, Environmental and Geo-Chemistry (AMGC), Vrije Universiteit Brussel, Brussels, Belgium.
authors.forename: Kim
authors.initials: K
authors.lastname: Croes
authors.affiliation: Laboratory of Plant Genetics (PLAN), Vrije Universiteit Brussel, Brussels, Belgium. Electronic address: Geert.Angenon@vub.ac.be.
authors.forename: Geert
authors.initials: G
authors.lastname: Angenon
mesh_terms: D000596:Amino Acids / Q000032:analysis*; D005561:Formates / Q000737:chemistry*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D010944:Plants / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D005561:Formates; C014667:methyl chloroformate
keywords: Amino acid profiling; Arabidopsis thaliana; Aspartate super pathway; GC–MS; Methyl chloroformate; Solid phase extraction