Protein bands from the SDS-PAGE gel after silver staining were destained and digested in gel with sequencing grade trypsin [trypsin (10 ng/μl) and 50 mM ammonium bicarbonate (pH 8.0)] overnight at 37°C. Peptides were extracted with 5% formic acid/50% acetonitrile and 0.1% formic acid/75% acetonitrile sequentially and then concentrated to ~20 μl. The extracted peptides were separated by an analytical capillary column (50 μm by 10 cm) packed with 5-μm spherical C18 reversed phase material (YMC, Kyoto, Japan). A Waters nanoACQUITY UPLC system (Waters, Milford, USA) was used to generate the following high-performance liquid chromatography gradient: 0 to 30% B in 60 min and 30 to 70% B in 15 min (A, 0.1% formic acid in water and B, 0.1% formic acid in acetonitrile). The eluted peptides were sprayed into a linear trap quadropole Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with a nano–electrospray ionization ion source. The mass spectrometer was operated in data-dependent mode with 1 mass spectrometry scan followed by 10 high-energy collisional dissociation tandem mass spectrometry scans for each cycle. Database searches were performed on an in-house Mascot server (Matrix Science Ltd., London, UK) against the International Protein Index human protein database.

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