Sample preparation and metabolite extraction:
Sample Extraction (For Indole acetate metabolites; IAA): 20 to 50mg of tissues was mixed with 1 ml cold extraction buffer and homogenised using a Mixer Mill MM301 bead mill at a frequency of 25 Hz for 5 min after adding 2 mm ceria-stabilised zirconium oxide beads. Add 1 μg of 13C indole acetate (or other suitable internal standards) and vortex well. Incubated this plant extracts at 4°C with continuous shaking (20 min) and centrifuge (15 min, 23 000 g at 4°C). Transfer the supernatant to a new vial and adjust pH to 2.7 with 1 M HCl
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 -80o C till further analysis or ship in dry ice.
7. Sample are reconstituted in mobile phase (or 50% methanol) before analysis
8. Processed Blank sample was prepared in the same manner as above where sample tissue is replaced with 50uL of buffer
9. Pooled QC sample: equal mix of all samples prepared after processing
Column: Kinetex C18 (100X 2.1; 2.6uM) at temperature 400C
Mobile phase A: 0.1 % acetic acid in Water
Mobile phase B: 0.1 % acetic acid in Methanol
Flow rate: 0.26 ml/min
Mobile phase Gradient:
Time/ %B: 0/5, 3/ 5, 10/75, 12/98, 16/98, 16.1/5, 19/5
Injection volume: 10 uL
ESI mode: Positive
Spray voltage 4.5 eV
Capillary Temp 3000C
Probe Heater Temp 3000C
Sheath Gas: 30 and Auxiliary Gas: 5 units
S-Lens RF Level 65v
m/z range: 60 to 900
1. Mobile phase (A:B; 90:10) blank: to check the background from LC column and LC-MS system
2. Processed Blank: to check the contamination and background from sample processing
3. Auxin standard mix: for the system suitability of the method
3. Pooled QC: 5 injections or till the LC-MS system gets stabilised
4. Plant tissue samples
5. Inject Pooled QC after each 6 tissue samples
7. Auxin standard mix: for the system suitability of the method
Thermo Xcalibur Quan software used for extracting peaks (as extracted ion chromatograms; EIC) for the metabolites of interest based on HRMS data with 5 ppm window for M+H adducts. The peaks were identified based on exact m/z and retention times in comparison with authentic standards. The peaks areas were used for relative quantification in the samples.
Targeted metabolites; chemical formulas:
Indole-3-acetic acid; C10H9NO2
Indole glucosinolate; C16H19N2O9S2
Indole-3-pyruvic acid; C11H9NO3
The MS/MS data for above metabolites can be downloaded from EMBL-MCF spectral library http://curatr.mcf.embl.de/MS2/