Protocol for abscisic acid (ABA) extraction from plant seeds
Jian You Wang, Lamis Berqdar, Salim Al-Babili
Disclaimer
The authors declare no competing interests.
Abstract
The plant hormone abscisic acid (ABA) regulates seed dormancy and germination. Here, we present a protocol for ABA extraction from plant seeds. We describe necessary steps required for preparation and extraction, followed by liquid chromatography-mass spectrometry (LC-MS/MS) analysis for ABA quantification.
Before start
To prepare before to start :
Prepare 2mL of working solution per sample: 100% Methanol spiked with 1 D6-ABA stock solution.
Attachments
Steps
Sample preparation
Grind 7-8 seeds in a Safe-Lock 2.0 mL Eppendorf tube with 3-4 beads for 0h 1m 0s – frequency 25-26 Hz.
Weight 100mg fine powder of ground seeds.
Add 1mL of Standard solution in each tube.
Sonicate the samples for 0h 15m 0s.
Centrifuge for 14000rpm, then transfer the supernatant to a new 2 mL Eppendorf tube.
Add another 1mL of Standard solution.
Sonicate 0h 15m 0s.
Centrifuge for 14000rpm.
Transfer the supernatant (2mL) to a 2 mL Eppendorf tube.
Dry the supernatant under vacuum by speedVac for 2h 0m 0s.
After evaporating the solvent, either keep the extracted samples at -20°C or proceed to the next step.
Re-dissolve the final extract in 120µL of acetonitrile : water [25:75 (v/v)] followed by 0h 1m 0s sonication.
Filter the re-suspended solution through a 0.22 µm filter into 0.1 mL micro-insert 29x5.7 mm clear glass tubes with inserted vials.
Tap the bottle to remove any bubbles.
Sample quantification
ABA quantification is performed by LC-MS/MS using a UHPLC-Triple-Stage Quadrupole Mass Spectrometer (Thermo Scientific Altis) machine.
Chromatographic separation is achieved on the Hypersil GOLD C18 Selectivity HPLC Columns (150 × 4.6 mm; 3 μm; fisher scientific) with mobile phases consisting of water (A) and acetonitrile (B), both containing 0.1% formic acid, and the following linear gradient (flow rate, 0.5 mL/min): 0–10 min, 15%–100 % B, follow it by washing with 100 % B for 0h 5m 0s and equilibration with 15 % B for 0h 2m 0s.
Inject 10µL of sample, maintain the column temperature at 35°C for each run.
Set the MS parameters of Thermo ScientificTM AltisTM as follows:
- negative mode,
- ion source of H-ESI,
- ion spray voltage of 3000 V,
- sheath gas of 40 arbitrary units,
- aux gas of 15 arbitrary units,
- sweep gas of 0 arbitrary units,
- ion transfer tube gas temperature of
350°C, - vaporizer temperature of
350°C, - collision energy of 20 eV,
- CID gas of 2 mTorr, and
- full width at half maximum (FWHM) 0.4 Da of Q1/Q3 mass.
