Quantitative Auxin Metabolite Profiling Using Stable Isotope Dilution UHPLC‐MS/MS

Ondřej Novák1, Aleš Pěnčík2, Ota Blahoušek3, Karin Ljung1

1 Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå, Sweden, 2 Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czech Republic, 3 Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany CAS, Faculty of Science, Palacký University, Olomouc, Czech Republic
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20028
Online Posting Date:  June, 2016
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Auxin, a plant hormone, is able to stimulate growth and to control developmental processes. To get a deeper level of understanding of how auxin activity is regulated in planta, we have developed a sensitive and selective mass spectrometry‐based protocol for multiplex quantification of indole 3‐acetic acid (IAA) as well as its precursors and degradation products. As part of the protocol development, we have also established a derivatization protocol for the IAA precursors IPyA and IAAld, compounds that are highly labile during sample extraction and purification. This article describes a standard isotope dilution method for quantitative profiling of the IAA metabolome in small amounts of plant tissues. © 2016 by John Wiley & Sons, Inc.

Keywords: auxin; IAA; LC‐MS/MS; metabolite profiling; SPE

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Table of Contents

  • Introduction
  • Basic Protocol 1: Purification of IAA and IAA Precursors, Conjugates, and Catabolites
  • Basic Protocol 2: Auxin Metabolite Profiling
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Purification of IAA and IAA Precursors, Conjugates, and Catabolites

  • Plant tissue samples (e.g., leaves, roots, hypocotyl, fruit)
  • Extraction buffer (see recipe)
  • Stable isotope‐labeled internal standards (IS; see recipe)
  • Derivatization reagent (see recipe)
  • 5%, 80%, and 100% (v/v) methanol (HPLC grade)
  • 5 mM and 3 M hydrochloric acid
  • Loading buffer (see recipe)
  • 1.5‐ml microcentrifuge tubes with safe‐lock lid
  • Scale, for weighing plant tissue
  • Liquid nitrogen
  • 2‐mm ceria‐stabilized zirconium oxide beads
  • Bead mill (e.g., Mixer Mill MM 301, Retsch GmbH)
  • Laboratory shaker
  • Refrigerated centrifuge
  • 1‐cc (30‐mg) Oasis HLB SPE columns (e.g., Waters)
  • Glass tubes
  • SPE Vacuum Manifold (e.g., 12‐port extraction plate)
  • Centrifuge vacuum concentrator (e.g., SpeedVac Concentrator)

Basic Protocol 2: Auxin Metabolite Profiling

  • Tissue samples (as prepared in protocol 1)
  • 10% and 100% (v/v) methanol (LC‐MS grade)
  • 0.1% (v/v) acetic acid in methanol (LC‐MS grade; mobile phase A)
  • 0.1% (v/v) acetic acid in water (mobile phase B)
  • Calibration solutions (see recipe)
  • Unlabeled and labeled auxin metabolite standards (see recipe)
  • Screw‐top autosampler vials (2‐ml) and glass inserts (250‐μl)
  • Ultrasonic bath
  • Reversed‐phase LC column (e.g., Kinetex C18 100 Å, 50 × 2.1 mm, 1.7 μm, Phenomenex)
  • UHPLC‐MS/MS system and associated equipment
  • Mass spectrometry software (e.g., Agilent MassHunter Workstation Software, Quantitative Analysis, version B.05.02)
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Literature Cited

Literature Cited
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