| Description |
Harvested sample is frozen by liquid N2 and ground to powder by a homogenizer, Shake Master (Biomedical Science, Tokyo, Japan). The powdered sample (100mg) are solved in 300uL 80% methanol solution. 10uL sample is injected into HPLC after 0.2um membrane filter treatment. HPLC conditions: Agilent 1100 series (Agilent), Column: TSKgel ODS-100V (4.6 x 250 mm, 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 45.0 min), 97% (45.1 to 50.0 min), 3% (50.1 to 57.0 min), Column temp.: 40 degree C, Flow rate=0.5mL/min, PDA: 200-650 nm (2 nm 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=102.09000, 102.13000, 103.11000, 116.14000, 121.12000, 130.16000, 158.00000, 171.15000, 202.13000, 235.18000, 249.21000, 252.22000, 294.26000, 308.00000, 376.04000, 379.04000, 380.03000, 540.61000, 540.95000, 609.28000, 810.41000, 1101.70000, 1123.68000, 1146.75000, 1147.76000. |