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Crushed and frozen samples (500 mg) were r … Crushed and frozen samples (500 mg) were resuspended in 1 mL of 50 mM phosphate buffer (pH 7.0), maintained on ice for 30 min, and then centrifuged (12,000 × g, 30 min, 4°C). The supernatant (400 μL) was mixed with 1.2 mL cold acetone and placed in a freezer at –30°C for 120 min. After centrifugation of the mixture (12,000 × g, 5 min, 4°C), the supernatant was carefully discarded and the pellet was suspended in 600 μL of water.
Performic acid reagent was prepared by mixing 30% (w/w) H2O2 and 98% (w/w) formic acid at a ratio of 1 to 9. The solution was mixed for 1 h at room temperature. The reagent (12.5 mL) was added to the sample solution (600 μL) and refrigerated at 4°C for 16 h. Most of the reagent was then removed under reduced pressure using a rotary evaporator (N-1000; EYELA, Tokyo, Japan). After evaporation, the sample was resuspended in 15 mL of 6 N HCl and then transferred to a screw-capped tube. The tube was placed in an oil bath (HOA-50A; ASONE, Tokyo, Japan) at 130°C for 20 h. The reagent was again removed under reduced pressure on a rotary evaporator and dissolved in 1 mL of MeOH. A total of 500 μL of this MeOH solution was used for CE-TOF MS analysis as described above. Total protein was quantified by the Bradford method. ein was quantified by the Bradford method.
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