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”.
   
5. 2 ml amber glass autosampler vials (Pacific lab, catalog number: SV11AW )
   
6. 250 μl inserts (Pacific lab, catalog number: IST0925N )
   
7. 1 L Schott bottles (acid washed)
   
8. Medicago truncatula seeds
   
9. Liquid N₂
   
10. Methanol (99.8%) HPLC grade (Thermo Fisher Scientific, ACROS Organics^TM, catalog number: 413770025 )
    
11. Propanol, HPLC grade (Fisher Scientific, catalog number: A461-212 )
    
12. Glacial acetic acid, HPLC grade (Fisher Scientific, catalog number: A113-50 )
    
13. Formic acid, HPLC grade (Fisher Scientific, catalog number: A117-50 )
    
14. Milli-Q water
    
15. Ultra high purity N₂ gas
    
16. Indole-2, 4, 5, 6, 7-d5-3-acetic acid, D5-IAA (Cambridge Isotope Laboratories, catalog number: DLM-2926 )
    Note: It is also named “Indole-3-acetic acid (indole-D5, 97-98%)” on Cambridge Isotope Laboratories website.
    
17. Indole-3-acetic acid, IAA (Sigma-Aldrich, catalog number: I3750 )
    
18. Indole-3-butyric acid, IBA (Sigma-Aldrich, catalog number: I5386 )
    
19. 4-chloro-indole-3-acetic acid, 4-Cl-IAA (OlChemIm Ltd., catalog number: 0031131 )
    
20. Phenylacetic acid, PAA (Sigma-Aldrich, catalog number: P16621 )
    
21. N-(3-indolylacetyl)-L-alanine, IAA-Ala (Sigma-Aldrich, catalog number: 345911 )
    
22. N-(3-indolylacetyl)-DL-aspartate, IAA-Asp (Sigma-Aldrich, catalog number: 345938 )
    
23. N-(3-indolylacetyl)-L-isoleucine, IAA-Ile (Sigma-Aldrich, catalog number: 347914 )
    Note: This product has been discontinued.
    
24. N-(3-indolylacetyl)-L-leucine, IALeu (OlChemIm Ltd., catalog number: 0031611 )
    
25. N-(3-Indolylacetyl)-L-phenylalanine, IAPhe (OlChemIm Ltd., catalog number: 0031623 )
    
26. N-(3-indolylacetyl)-L-tryptophan, IATrp (OlChemIm Ltd., catalog number: 0031631 )
    
27. N-(3-indolylacetyl)-L-valine, IAVal (OlChemIm Ltd., catalog number: 0031641 ) 
    Notes:
1. Solvents and chemicals should be of the highest available purity.
   
2. Auxin standards and internal standards are prepared in HPLC grade methanol. 1,000 mg/L stock solutions are prepared using acid washed volumetric flasks and aliquoted (1 ml) into 2 ml amber glass vials with crimped caps and stored at -80 °C until use.

Equipment

1. TissueLyser LT (QIAGEN, catalog number: 69980 )
   
2. Sonicator bath (Cole-Parmer Instrument Company, model: 8545-4 )
   
3. SpeedVac vacuum centrifuge (LabConco)
   
4. Agilent 6530 High Resolution Accurate Mass LC-MS Q-TOF with Agilent Jetstream (AJS) ESI ion source interface
   
5. Agilent Zorbax Eclipse high resolution XDB-C18 2.1 x 50 mm, 1.8 μm LC column

Software

1. Agilent MassHunter software version B.05.00 for data acquisition and data analysis

Procedure