| MS Description
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Harvested sample is frozen by liquid N2 an … 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). i Dep MS/MS 5th most intense ion from (1).
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