| MS Description
|
The samples were cut into small pieces and … The samples were cut into small pieces and then frozen in liquid N2 and resulting powder (100mg) are solved in 300uL 100% methanol solution and homogenized with zirconia beads in a TissueLyser II. The supernatant was filtered using a Mono-Spin C18 column and filtered through a 0.2 µm polyvinylidene difluoride (PVDF) membrane. 2µL of sample is injected into HPLC. HPLC conditions: Ultimate 3000 RSLC (Thermo Fisher Scientific Inc.), Column: InertSustain AQ-C18 (column size: 2.1 × 150 mm; particle size: 3.0 μm; GL Science Inc.), Solvent: A; 0.1% formic acid in water, B; 0.1% formic acid in acetonitrile, Gradient: 2% B for 0 to 3 min, 2 to 98% B for 3 to 30 min, 98% B for 30 to 35 min, and 2% B for 35 to 40 min. Column temp.: 40 degree C, Flow rate=0.2mL/min, PDA: 190-950 nm (2 nm step). Orbitrap-MS conditions: Q Exactive Mass Spectrometer (Thermo Fisher Scientific), ESI positive mode, spray voltage: 3.2 kV, capillary temperature: 300 degree C, Full mass scan condition (scan range: m/z 80–1200, resolution: 70000 at m/z 200, AGC target: 1e6), MS/MS conditoin: up to 10 data-dependent HCD MS2 scans (AGC target: 1e5, max IT: 50 ms, resolution: 17500 at m/z 200, isolation window: m/z 2.0, stepped (N)CE: 10/50/80, underfill ratio: 5%, and dynamic exclusion: 20 s). l ratio: 5%, and dynamic exclusion: 20 s).
|
