Analysis and Quantification of Glucosinolates

Christoph Crocoll1, Barbara Ann Halkier1, Meike Burow1

1 DynaMo Center, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Copenhagen, Denmark
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20027
Online Posting Date:  June, 2016
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Abstract

Recent advances in liquid chromatography and mass spectrometry have made it possible to increase the throughput of phytochemical analyses at high sensitivity. These improvements have made it more feasible to monitor metabolic processes at the metabolite level. Glucosinolates, the primary defense compounds in the model plant Arabidopsis thaliana, in particular, have gained increasing attention as model compounds for quantitative genetics and metabolic regulation. Depending on the plant species, tissue, glucosinolate content, complexity of the glucosinolate profile, and, most importantly, the overall purpose of the experiment, different choices need to be made regarding the methods of extraction and analysis. In this chapter, we describe different approaches for the analysis of glucosinolates and highlight advantages, disadvantages, and technical pitfalls. © 2016 by John Wiley & Sons, Inc.

Keywords: chromatography; glucosinolates; metabolite profiling; mass spectrometry; plant specialized metabolism

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Extraction and Analysis of dsGLS by HPLC‐UV/Diode Array Detector (DAD)
  • Support Protocol 1: Preparative GLS Extraction
  • Support Protocol 2: Sulfatase Purification for Sulfatase Solution
  • Alternate Protocol 1: Large‐Volume Extraction for Analysis of dsGLS
  • Alternate Protocol 2: Analysis of dsGLS by LC‐MS/TQ
  • Support Protocol 3: Identification and Optimization of MRM Transitions of Intact GLS and dsGLS
  • Basic Protocol 2: Extraction and Analysis of Intact GLS by LC‐MS/qTOF
  • Alternate Protocol 3: Analysis of Intact GLS by LC‐MS/TQ
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Extraction and Analysis of dsGLS by HPLC‐UV/Diode Array Detector (DAD)

  Materials
  • Fresh or freeze‐dried plant material
  • 85% (containing internal standard; see recipe) and 70% (v/v) methanol
  • DEAE‐Sephadex A‐25 (GE Healthcare, cat. no. 17‐0170‐02)
  • Sulfatase solution ( protocol 3)
  • Acetonitrile (LC‐MS Chromasolv; Fluka, cat. no. 34967)
  • Chrome or stainless steel ball bearings, 3‐mm‐diameter (e.g., VXB, cat. no. KIT12065, or any other supplier of ball bearings)
  • 1.2‐ml microelution tubes, strips of 8 (Deltalab, cat. no. 408002)
  • Mixer Mill 303 (Retsch; alternatively a paint shaker can be used)
  • 96‐well filter plates, 0.45 µm pore size (MultiScreenHTS; EMD Millipore, cat no. MSHVN 4550)
  • Multiscreen column loader (Millipore, MACL 09645)
  • Centrifuge with plate adapter
  • Vacuum manifold
  • 96‐well plate
  • HPLC equipped with a DAD or UV detector (e.g., Agilent HP1200 Series)
  • C18 reversed‐phase column [Zorbax SB‐Aq, 25 cm × 4.6 mm, 5 µm particle size (Agilent) or Lichrospher RP18‐5, 25 cm × 4.6 mm, 5 µm particle size (Supelco)]

Support Protocol 1: Preparative GLS Extraction

  Materials
  • Seeds (e.g., from Sinapis alba, Lepidium sativum, Brassica nigra, Isatis tinctoria)
  • 80% (v/v) methanol
  • 10% DEAE‐Sephadex suspension (see recipe)
  • 3:2:5 formic acid/isopropanol/water
  • 0.5 M K 2SO 4/3% (v/v) isopropanol
  • Ethanol, absolute (>96%)
  • Supelcosil LC18DB hydrophobic column, 5‐µm particle size; 10 × 250 mm
  • Trifluoroacetic acid (TFA)
  • Acetonitrile (LC‐MS Chromasolv, Fluka, cat. no. 34967)
  • 15‐ml conical screw‐cap tubes
  • Coffee mill or Ultra‐Turrax homogenizer (IKA)
  • Centrifuge
  • Empty chromatography columns, 1.5 × 12 cm (Econo‐Pac Chromatography Columns, BioRad, cat. no. 7321010)
  • SpeedVac or rotary evaporator
  • HPLC equipped with a DAD or UV detector (e.g., Agilent HP1200 Series)
  • Additional reagents and equipment for HPLC‐DAD ( protocol 1)

Support Protocol 2: Sulfatase Purification for Sulfatase Solution

  Materials
  • Sulfatase from Helix pomatia (Sulfatase Type H1, Sigma, cat. no. S9626, ≥10,000 U/g solid)
  • Ethanol, absolute (>96%)
  • 20 mM sodium acetate buffer, pH 5.0
  • 15‐ml conical screw‐cap tubes
  • 1.5‐ml microcentrifuge tubes

Alternate Protocol 1: Large‐Volume Extraction for Analysis of dsGLS

  Materials
  • Fresh or freeze‐dried plant material
  • 85%, 70%, and 60% (v/v) methanol
  • GLS standard for quantification (see protocol 2)
  • 10% (w/v) DEAE‐Sephadex suspension (see recipe)
  • Sulfatase solution ( protocol 3)
  • 15‐ml conical screw cap tubes
  • 2‐ml microcentrifuge tubes
  • Chrome or stainless steel ball bearings, 3‐mm‐diameter (e.g., VXB, cat. no. KIT12065, or any other supplier of ball bearings) or Ultra‐Turrax homogenizer (IKA)
  • Empty chromatography columns (Poly‐Prep chromatography columns; BioRad, cat. no. 7311550)
  • 50ºC heat block or SpeedVac evaporator

Alternate Protocol 2: Analysis of dsGLS by LC‐MS/TQ

  Materials
  • Extracted dsGLS ( protocol 1, steps 1 to 11)
  • Acetonitrile (LC‐MS Chromasolv, Fluka, cat. no. 34967)
  • 0.05% formic acid (Sigma‐Aldrich, cat. no. F0507, reagent grade) in MilliQ‐grade water
  • 0.05% formic acid (Sigma‐Aldrich, cat. no. F0507, reagent grade) in LC‐MS‐grade acetonitrile (LC‐MS Chromasolv; Fluka, cat. no. 34967)
  • UHPLC‐MS/TQ instrument
  • Kinetex 1.7 μ XB‐C18 column (100 × 2.1 mm, 1.7 µm, 100 Å, Phenomenex)
NOTE: The response factors for quantification by MRM assay depend on the individual settings for ionization and fragmentation and therefore can not be transferred between instruments from different manufacturers or of different types.

Support Protocol 3: Identification and Optimization of MRM Transitions of Intact GLS and dsGLS

  Materials
  • Pure intact GLS or dsGLS or mixtures thereof at concentrations of 1 to 10 µM
  • MS/TQ instrument
  • Syringe pump for direct infusion

Basic Protocol 2: Extraction and Analysis of Intact GLS by LC‐MS/qTOF

  Materials
  • Extracted dsGLS ( protocol 1, steps 1 to 4)
  • 0.05% formic acid (Sigma‐Aldrich, cat. no. F0507, reagent grade) in MilliQ‐grade water
  • 0.05% formic acid (Sigma‐Aldrich, cat. no. F0507, reagent grade) in LC‐MS‐grade acetonitrile (LC‐MS Chromasolv, Fluka, cat. no. 34967)
  • Kinetex 1.7 μ XB‐C18 UHPLC column (100 × 2.1 mm, 1.7 µm, 100 Å, Phenomenex)
  • 96‐well filter‐plates, 0.22‐µm pore size
  • UHPLC‐MS/qTOF instrument

Alternate Protocol 3: Analysis of Intact GLS by LC‐MS/TQ

  Materials
  • Plant extract ( protocol 1, steps 1 to 4)
  • 0.05% formic acid (Sigma‐Aldrich, cat. no. F0507, reagent grade) in MilliQ‐grade water
  • 0.05% formic acid (Sigma‐Aldrich, cat. no. F0507, reagent grade) in LC‐MS‐grade acetonitrile (LC‐MS Chromasolv, Fluka, cat. no. 34967)
  • Kinetex 1.7 μ XB‐C18 UHPLC column (100 × 2.1 mm, 1.7 µm, 100 Å, Phenomenex)
  • 96‐well filter‐plates, 0.22 µm pore size
  • UHPLC‐MS/qTOF instrument
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Figures

Videos

Literature Cited

Literature Cited
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