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Harvested sample is frozen by liquid N2 an … Harvested sample is frozen by liquid N2 and resulting powder (100mg) are solved in 500uL 80% methanol solution. Cells are homogenized by TissueLyser (QUIAGEN) and centrifuged (15000rpm, 10min, 4 degree C). 20uL of the supernatant (300uL) is injected into HPLC after 0.2um membrane filter treatment. HPLC conditions: Agilent 1100 series (Agilent), Column: TSKgel Amide-80 (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);80 to 45% (0.0 to 30.0 min), 45 to 35% (30.0 to 30.1 min), 35% (30.1 to 35.0 min), 35% to 80% (35.0 to 35.1 min), 80% (35.1 to 42.0 min), Column temp.: 40 degree C, Flow rate=0.8mL/min, PDA: 200-600 nm (2 nm step). Orbitrap-MS conditions: Filter 1: FTMS + c norm !corona !pi res=60000 o(150.0-2000.0); 2: FTMS + c norm !corona !pi res=7500 Dep MS/MS Most intense ion from (1); 3: FTMS + c norm !corona !pi res=7500 Dep MS/MS 2nd most intense ion from (1). 0 Dep MS/MS 2nd most intense ion from (1).
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