Protein analysis by LC-MS/MS. The enzymatically digested protein fragments were applied to a liquid chromatograph (EASY-nLC 1000; Thermo Fisher Scientific Inc.) coupled to a Q Exactive hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific Inc.) with a nanospray ion source in positive mode. The peptides derived from protein fragments were separated on a nano–high-performance liquid chromatography capillary column C18 (0.075 mm inside diameter × 150 mm length, 3 mm particle size, Nikkyo Technos Co. Ltd.). The mobile phase “A” was water with 0.1% formic acid, and the mobile phase “B” was acetonitrile with 0.1% formic acid. Two different slopes were used for a 60-min gradient at a flow rate of 300 ml/min: 5 to 35% B in 48 min, and then 35 to 65% B in 12 min. The Q Exactive MS was operated in top 10 data-dependent scan mode. The parameters of Q Exactive were as follows: spray voltage, 2.3 kV; capillary temperature, 275°C; mass range, 350 to 1800 m/z (mass/charge ratio); normalized collision energy, 28%. Raw data were acquired with Xcalibur software.

Protein identification. The MS and MS/MS data were searched against the Swiss-Prot database using Proteome Discoverer (Thermo Fisher Scientific Inc.) with the MASCOT search engine software (Matrix Science Inc.). The search parameters were as follows: enzyme, Lys-C protease static modifications, carbamidomethyl (Cys); dynamic modifications, oxidation (Met); precursor mass tolerance, ± 6 ppm (parts per million); fragment mass tolerance, ± 20 mDa; maximum missed cleavages, 1. The proteins were considered identified when their false discovery rates were less than 5%.

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