6. Micro glass insert (VWR 28 Â 5.6 mm, or equivalent).
7. Acquity UPLC BEH C 18 column (100 Â 2.1 mm; 1.7 μm;
Waters) with an Acquity UPLC BEH C 18 guard column
(5 Â 2.1 mm; 1.7 μm; Waters), or equivalent.
8. Dionex UltiMate 3000 UHPLC system (Thermo Scientific) or
equivalent.
9. Heated-electrospray Ionization-Hybrid Quadrupole-Orbitrap
Mass Spectrometer (HESI-Q-Orbitrap MS, Thermo Scientific
Q Exactive Plus) or equivalent.
3 Methods
All steps used for extraction and derivatization of DIALs should be
carried out under dim light, unless otherwise specified. This
method has been optimized for detecting DIALs in Arabidopsis
thaliana. Detecting DIALs in other plant species or tissues might
require additional optimization.
3.1 Plant Material
Preparation
1. Collect fresh Arabidopsis seedlings (10-day) in 2 mL microcentrifuge tubes and keep them frozen in liquid nitrogen for
5 min (see Note 6).
2. Transfer tubes with plant material to the freeze-dry system and
dry the material for 12 h (see Note 7).
3. Add 3 beads in each tube after drying. Then transfer tubes in
liquid nitrogen for 1 min (see Note 8).
4. Transfer tubes to the bead beater and homogenize plant material for 1 min.
5. Weigh between 25 and 30 mg dry weight plant material in a
2 mL microcentrifuge tube. Work quickly and cover the
weighed material with aluminum foil. Note the exact weight.
3.2 Crude DIALs
Extraction
1. Prepare the extraction solution by adding 10 mg of BHT to a
volumetric flask and make up to 10 mL with acetonitrile. Keep
the extraction solution on ice.
2. Add 0.5 mL of extraction solution to each tube and mixture
gently. Transfer tubes to an ultrasonic bath for the extraction
for 15 min at 25
C. After that, transfer tubes to a precooled
microcentrifuge at 18,000 Â g for 5 min at 4
C. Collect the
supernatant and reextract the pellet with 0.5 mL of extraction
solution. Combine the two supernatants and dry it using a
concentrator (see Note 9).
Method for Identification of Carotenoid-Derived Dialdehydes
181
7. Acquity UPLC BEH C 18 column (100 Â 2.1 mm; 1.7 μm;
Waters) with an Acquity UPLC BEH C 18 guard column
(5 Â 2.1 mm; 1.7 μm; Waters), or equivalent.
8. Dionex UltiMate 3000 UHPLC system (Thermo Scientific) or
equivalent.
9. Heated-electrospray Ionization-Hybrid Quadrupole-Orbitrap
Mass Spectrometer (HESI-Q-Orbitrap MS, Thermo Scientific
Q Exactive Plus) or equivalent.
3 Methods
All steps used for extraction and derivatization of DIALs should be
carried out under dim light, unless otherwise specified. This
method has been optimized for detecting DIALs in Arabidopsis
thaliana. Detecting DIALs in other plant species or tissues might
require additional optimization.
3.1 Plant Material
Preparation
1. Collect fresh Arabidopsis seedlings (10-day) in 2 mL microcentrifuge tubes and keep them frozen in liquid nitrogen for
5 min (see Note 6).
2. Transfer tubes with plant material to the freeze-dry system and
dry the material for 12 h (see Note 7).
3. Add 3 beads in each tube after drying. Then transfer tubes in
liquid nitrogen for 1 min (see Note 8).
4. Transfer tubes to the bead beater and homogenize plant material for 1 min.
5. Weigh between 25 and 30 mg dry weight plant material in a
2 mL microcentrifuge tube. Work quickly and cover the
weighed material with aluminum foil. Note the exact weight.
3.2 Crude DIALs
Extraction
1. Prepare the extraction solution by adding 10 mg of BHT to a
volumetric flask and make up to 10 mL with acetonitrile. Keep
the extraction solution on ice.
2. Add 0.5 mL of extraction solution to each tube and mixture
gently. Transfer tubes to an ultrasonic bath for the extraction
for 15 min at 25
C. After that, transfer tubes to a precooled
microcentrifuge at 18,000 Â g for 5 min at 4
C. Collect the
supernatant and reextract the pellet with 0.5 mL of extraction
solution. Combine the two supernatants and dry it using a
concentrator (see Note 9).
Method for Identification of Carotenoid-Derived Dialdehydes
181