Mass Spectrometry Imaging of Metabolites in Barley Grain Tissues

Manuela Peukert1, Wai Li Lim2, Udo Seiffert3, Andrea Matros4

1 Cluster of Excellence on Plant Sciences (CEPLAS), University of Cologne, Cologne, 2 Australian Research Council Centre of Excellence in Plant Cell Walls (ARC CoE), University of Adelaide, Urrbrae, 3 Biosystems Engineering, Fraunhofer Institute for Factory Operation and Automation IFF, Magdeburg, 4 Leibniz‐Institute of Plant Genetics and Crop Plant Research (IPK), Applied Biochemistry Group, Gatersleben
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20037
Online Posting Date:  December, 2016
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Higher plants are composed of a multitude of tissues with particular functions, reflected by distinct profiles of transcripts, proteins, and metabolites. Although the rapid development of “omics” technologies has advanced plant science tremendously within recent years, analysis is frequently performed on whole organ or whole plant extracts, causing the loss of spatial information. Mass spectrometry–based imaging (MSI) approaches have become a powerful tool to decipher spatially resolved molecular information. Matrix‐assisted laser desorption/ionization (MALDI) is the most widespread ionization method utilized for MSI and has recently been applied to plant science. A range of different plant organs and tissues has been successfully analyzed by MSI, and patterns of various classes of metabolites from primary and secondary metabolism have been obtained. This protocol describes a method for analysis of spatial metabolite distributions in cryosections of developing barley grains. Detailed procedures for sample preparation, mass spectrometry measurement, and data analysis are provided. © 2016 by John Wiley & Sons, Inc.

Keywords: barley grain; mass spectrometry imaging (MSI); metabolite distribution; MALDI‐MS imaging; on‐tissue digestion

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Table of Contents

  • Introduction
  • Basic Protocol 1: Cryosectioning of Developing Barley Grains
  • Basic Protocol 2: Matrix Application and Slide Preparation
  • Basic Protocol 3: MALDI‐MS Measurement of Small Molecules
  • Basic Protocol 4: Directed Data Analysis
  • Basic Protocol 5: Undirected Data Analysis
  • Support Protocol 1: Structural Analysis of Oligomeric Sugars by Invertase Digestion
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Cryosectioning of Developing Barley Grains

  • Developing barley grains
  • Liquid nitrogen
  • Optimal cutting temperature (OCT) medium
  • White correction fluid (e.g., Wite‐Out)
  • Cryotome, e.g., Leica CM3050 (Leica Microsystems)
  • Indium tin oxide (ITO)–coated glass slides (Bruker Daltonics)
  • Brush, forceps, razor blade
  • Desiccator and vacuum pump
  • Stereomicroscope (e.g., Leica MZ6) and digital camera (e.g., AxioCam ICc1, Zeiss) or similar microscopic image acquisition system

Basic Protocol 2: Matrix Application and Slide Preparation

  • Sample sections (see protocol 1 or protocol 6Support Protocol)
  • 2,5‐Dihydroxybenzoic acid (DHB, Sigma‐Aldrich)
  • Methanol, MS‐grade
  • Water, MS‐grade
  • Trifluoroacetic acid (TFA, >99%, Sigma‐Aldrich)
  • Sonication bath
  • ImagePrep device (Bruker Daltonics)

Basic Protocol 3: MALDI‐MS Measurement of Small Molecules

  • Sample sections with matrix deposited (see protocol 2)
  • Isopropanol
  • Low‐lint cleaning wipes
  • ultrafleXtreme MALDI‐TOF mass spectrometer (Bruker Daltonics)
  • FlexControl and flexImaging software (Bruker Daltonics)
  • MTP Slide‐Adapter II (Bruker Daltonics)
  • Calibration solution: 1:1 mixture of polyethylene glycol (PEG) 200 and 600, diluted 1:300 in 30% (v/v) acetonitrile with 0.1 % (v/v) trifluoroacetic acid (TFA)

Basic Protocol 4: Directed Data Analysis

  • Yeast invertase (Sigma‐Aldrich)
  • 20 mM KH 2PO 4 (Sigma‐Aldrich)
  • 20 mM K 2HPO 4 (Sigma‐Aldrich)
  • Water, MS‐grade
  • Methanol, MS‐grade
  • Coverslips
  • ImagePrep device (Bruker Daltonics)
  • Moist chamber
  • 55°C water bath
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Literature Cited

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