Metabolite extraction from plant tissue

Protocol/MCF/SamplePrep/03: Metabolite extraction from plant tissues (Novák et al., 2016)

Sample required:Plant material required: 20-50 mg finely homogenized tissue

Materials and preparations: MS grade Methanol (with suitable IS mix 100 ng), MilliQ Water, with suitable internal standards mix 100 ng, HLB (30 mg) SPE columns, SPE assembly, 5% Methanol, 80% methanol Extraction buffer (50 mM sodium phosphate buffer, pH 7.0, containing 0.1%diethyldithiocarbamate)Loading bufferUse 1 ml of the extraction buffer to test the amount of 1M HCl needed for itsacidification to pH 2.7. Prepare the loading buffer by adding twice the amount of1 M HCl per milliliter of extraction buffer. (Addition of 0.5 ml sample extract to 0.5 ml loading buffer must give a pH 2.7)

Note: Keep diluents in chilled condition (0-4oC)

Sample preparation and metabolite extraction:

1. Sample Extraction (For Indole acetate metabolites; IAA): 20 to 50 mg of tissues was mixed with 1 ml cold extraction buffer and homogenized using a Mixer Mill MM301 bead mill (Retsch GmbH (http://www.retsch.com)) at a frequency of 25 Hz for 5 min after adding 2 mm ceria-stabilized zirconium oxide beads.
2. Add 1 μg of 13C indole acetate (or other suitable internal standards) and vortex well.
3. Incubated this plant extracts at 4°C with continuous shaking (20 min) and centrifuge (15 min, 23 000 g at 4°C)
4. Transfer the supernatant to a new vial and adjust pH to 2.7 with 1 M HCl
5. SPE procedure: a. Prepare SPE vacuum assembly with HLB (30mg) column cartridges.
   b. Condition SPE column with 1 mL of methanol and 1mL of water c. Equilibrate the column with 250 μl of 5 mM HCl. (Do not let column dry) d. Load equilibrate column with 0.5 ml loading buffer e. Load 0.5 ml Sample onto the SPE column, and mix intensely with the loading buffer. Pass the mixture slowly through the HLB sorbent immediately. f. Wash the column with 2 ml of 5% methanol. g. Put clean glass tubes into the manifold rack, and then elute the sample from the column with 2 ml of 80% methanol. h. Collect sample eluent and evaporate the samples to dryness in a SpeedVac concentrator or under nitrogen stream at room temperature.

6. Store samples at -80°C till further analysis or ship in dry ice.

7. Sample are reconstituted in mobile phase (or 50% methanol) before analysis

References

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1. Novák, O., Pěnčík, A., Blahoušek, O., & Ljung, K. (2016). Quantitative auxin metabolite profiling using stable isotope dilution UHPLC‐ms/MS. Current Protocols in Plant Biology, 1(2), 419–430. https://doi.org/10.1002/cppb.20028