| MS Description
|
<Blank preparation>
80% methanol was … <Blank preparation>
80% methanol was prepared from methanol (Optima LC/MS, Fisher Chemical) and water (Optima LC/MS, Fisher Chemical) and cleaned up with a disc filter (millex-LG, 0.20um, MILLIPORE).
<Metabolite extraction>
The metabolites were extracted with 80% methanol (see "Blank preparation") as follows: 1) Measure about 10mg of plant sample in 1.5ml microtube. 2) Ground the sample in the tube with pestle. 3) Add 20 times the volume of cool 80% methanol and mixed well. 4) Incubate it on ice for 30min and then centrifuge it at 15,000/g for 20 min at 4 degree. 5) Collect the supernatants in a new glass vial. 6) Repeat steps 3 to 5 once. 7) Combine all the supernatants in same glass vial. 8) Clean up the solution with the disc filter (millex-LG, 0.20um, MILLIPORE).
<LC-Orbitrap-MS analysis>
A 10ul sample is injected into HPLC. HPLC conditions: Agilent 1100 series (Agilent), Column: TSKgel ODS-100V (4.6 x 250mm, 5 micrometer; TOSOH), Solvent: A; 0.1% formic acid aq. B; ACN (addition 0.1% formic acid fc.), Gradient: (B);3 to 97% (0.0 to 45.0min), 97% (45.1 to 50.0min), 3% (50.1 to 57.0min), Column temp.: 40 degree C, Flow rate=0.5mL/min, PDA: 190-950nm (2nm step). Orbitrap-MS conditions: Filter 1: FTMS + c norm !corona !pi res=60000 o(100.0-1500.0); 2: ITMS + c norm !corona !pi Dep MS/MS Most intense ion from (1); 3: ITMS + c norm !corona !pi Dep MS/MS 2nd most intense ion from (1); 4: ITMS + c norm !corona !pi Dep MS/MS 3rd most intense ion from (1); 5: ITMS + c norm !corona !pi Dep MS/MS 4th most intense ion from (1); 6: ITMS + c norm !corona !pi Dep MS/MS 5th most intense ion from (1)., Rejected mass=235.00000, 376.00000, 609.00000, 810.00000, 1123.00000. 6.00000, 609.00000, 810.00000, 1123.00000.
|
