TITLE: Protocol for abscisic acid (ABA) extraction from plant seeds
AUTHORS: Jian You Wang, Lamis Berqdar, Salim Al-Babili

[DESCRIPTION]
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 2 mL of working solution per sample: 100% Methanol spiked with 1 1800 D6-ABA stock solution.

[GUIDELINES]
Author Contributions

S.A.-B. and J.Y.W. conceived the project. J.Y.W., and L.B. conducted experiments. 
L.B., J.Y.W., and S. A.-B. wrote, reviewed, and edited the protocol. 

Competing interests

The authors declare no competing interests.

[STEPS]
SECTION: Sample preparation
1. Grind 7-8 seeds in a Safe-Lock 2.0 mL Eppendorf tube with 3-4 beads for 1 min – frequency 25-26 Hz.
SECTION: Sample preparation
2. Weight 100 mg fine powder of ground seeds.

SECTION: Sample preparation
3. Add 1 mL of Standard solution in each tube.

SECTION: Sample preparation
4. Sonicate the samples for 15 min.

SECTION: Sample preparation
5. Centrifuge for 14000 rpm, 5 min, then transfer the supernatant to a new 2 mL Eppendorf tube.
SECTION: Sample preparation
6. Add another 1 mL of Standard solution.

SECTION: Sample preparation
7. Sonicate 15 min.

SECTION: Sample preparation
8. Centrifuge for 14000 rpm, 5 min.

SECTION: Sample preparation
9. Transfer the supernatant (2 mL) to a 2 mL Eppendorf tube.

SECTION: Sample preparation
10. Dry the supernatant under vacuum by speedVac for 120 min.

SECTION: Sample preparation
11. After evaporating the solvent, either keep the extracted samples at -20 °C or proceed to the next step.
SECTION: Sample preparation
12. Re-dissolve the final extract in 120 µL of acetonitrile : water [25:75 (v/v)] followed by 1 min sonication.

SECTION: Sample preparation
13. Filter the re-suspended solution through a   filter into 0.1 mL micro-insert 29x5.7 mm clear glass tubes with inserted vials.
SECTION: Sample preparation
14. Tap the bottle to remove any bubbles.
SECTION: Sample quantification
15. ABA quantification is performed by LC-MS/MS using a UHPLC-Triple-Stage Quadrupole Mass Spectrometer (Thermo Scientific Altis) machine.
SECTION: Sample quantification
16. 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 5 min and equilibration with 15 % B for 2 min.
SECTION: Sample quantification
17. Inject 10 µL of sample, maintain the column temperature at 35 °C for each run.
SECTION: Sample quantification
18. 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.