http://www.bio-protocol.org/e1843 Vol 6, Iss 12, Jun 20, 2016 Copyright © 2016 The Authors; exclusive licensee Bio-protocol LLC. 1 Quantifying Auxin Metabolites in Young Root Tissue of Medicago truncatula by Liquid Chromatography Electrospray-ionisation Quadrupole Time-of-flight (LC-ESI-QTOF) Tandem Mass Spectrometry Jason Liang Pin Ng 1 , Thy T. Truong 2 , Charles H. Hocart 2 and Ulrike Mathesius 1* 1 Division of Plant Science, Research School of Biology, The Australian National University, Canberra, Australia; 2 Mass Spectrometry Facility, Research School of Biology, The Australian National University, Canberra, Australia * For correspondence: Ulrike.Mathesius@anu.edu.au [Abstract] Auxins represent a major group of phytohormones controlling plant development. The spatio-temporal regulation of auxin gradients is essential for the initiation, growth and correct development of plant organs. Because auxins and their metabolites occur at trace levels in plant tissue, experiments requiring identification plus their selective and specific quantification can be most conveniently achieved using mass spectrometry (MS) and the associated chromatographic methods. With the advent of appropriate liquid-based ionisation techniques, emphasis has moved from the use of gas chromatography as the sample interface to the MS (GC/MS), with its concomitant need for derivatisation, to the more sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS). We describe an optimized liquid chromatography electrospray-ionisation quadrupole time-of-flight (LC-ESI-QTOF) methodology for the quantification of auxins. While the solvent extraction of young Medicago truncatula (M. truncatula) roots, as described herein, is relatively straightforward, older, woody or oily plant tissues may also be analyzed with appropriate modification to remove interferences and/or enhance extraction efficiency. In our hands, the analytical assay has proved sufficiently sensitive for the quantification of auxins to investigate their roles in various organogenic events, such as root nodulation in M. truncatula. Further increases in sensitivity can be expected with the use of the latest generation of instruments. Materials and Reagents 1. Stainless steel balls, 3 mm diameter, sequentially washed with detergent, rinsed with tap water, Milli-Q water, and then sterilized in ethanol overnight prior to being air dried before use (AussieSapphire) 2. 2 ml sterile non-autoclaved Eppendorf tubes (Sigma-Aldrich, catalog number: T2795) 3. 1.5 ml sterile non-autoclaved microtubes (Sigma-Aldrich, catalog number: SIAL311NZ1.5C) 4. Nanosep ® MF GHP 0.45 μm filter (Pall Life Sciences, catalog number: ODGHPC35) Note: Currently, it is “VWR International, catalog number: ODGHPC35”.