title: Analysis of plant nucleotide sugars by hydrophilic interaction liquid chromatography and tandem mass spectrometry.
abstract: Understanding the intricate metabolic processes involved in plant cell wall biosynthesis is limited by difficulties in performing sensitive quantification of many involved compounds. Hydrophilic interaction liquid chromatography is a useful technique for the analysis of hydrophilic metabolites from complex biological extracts and forms the basis of this method to quantify plant cell wall precursors. A zwitterionic silica-based stationary phase has been used to separate hydrophilic nucleotide sugars involved in cell wall biosynthesis from milligram amounts of leaf tissue. A tandem mass spectrometry operating in selected reaction monitoring mode was used to quantify nucleotide sugars. This method was highly repeatable and quantified 12 nucleotide sugars at low femtomole quantities, with linear responses up to four orders of magnitude to several 100pmol. The method was also successfully applied to the analysis of purified leaf extracts from two model plant species with variations in their cell wall sugar compositions and indicated significant differences in the levels of 6 out of 12 nucleotide sugars. The plant nucleotide sugar extraction procedure was demonstrated to have good recovery rates with minimal matrix effects. The approach results in a significant improvement in sensitivity when applied to plant samples over currently employed techniques.
authors.affiliation: Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Ito
authors.affiliation: Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm 14476, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Herter
authors.affiliation: Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Edward E K
authors.initials: EE
authors.lastname: Baidoo
authors.affiliation: Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Jeemeng
authors.initials: J
authors.lastname: Lao
authors.affiliation: Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Miguel E
authors.initials: ME
authors.lastname: Vega-Sánchez
authors.affiliation: Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: A
authors.initials: A
authors.lastname: Michelle Smith-Moritz
authors.affiliation: Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Department of Bioengineering, University of California, Berkeley, CA 94720, USA.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Adams
authors.affiliation: Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Department of Bioengineering, University of California, Berkeley, CA 94720, USA; Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720, USA.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.affiliation: Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm 14476, Germany; RWTH Aachen University, Institute for Biology I, Aachen 52056, Germany; Forschungszentrum Jülich, IBG-2: Plant Sciences, Jülich 52425, Germany.
authors.forename: Björn
authors.initials: B
authors.lastname: Usadel
authors.affiliation: Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Petzold
authors.affiliation: Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. Electronic address: jlheazlewood@lbl.gov.
authors.forename: Joshua L
authors.initials: JL
authors.lastname: Heazlewood
mesh_terms: D017360:Arabidopsis / Q000737:chemistry / Q000378:metabolism; D002241:Carbohydrates / Q000032:analysis* / Q000737:chemistry; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D057927:Hydrophobic and Hydrophilic Interactions; D009711:Nucleotides / Q000032:analysis*; D012275:Oryza / Q000737:chemistry / Q000378:metabolism; D018515:Plant Leaves / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D002241:Carbohydrates; D009711:Nucleotides
keywords: Arabidopsis; Hydrophilic interaction liquid chromatography; Nucleotide sugars; Plant cell walls; Rice; Selected reaction monitoring