Proteins were separated by SDS–polyacrylamide gel electrophoresis, and the gel bands of interest were excised, reduced with 5 mM dithiotreitol, and alkylated with 11 mM iodoacetamide. In-gel digestion was then carried out with sequencing grade modified trypsin in 50 mM ammonium bicarbonate at 37°C overnight. The peptides were extracted twice with 0.1% trifluoroacetic acid (TFA) in 50% acetonitrile aqueous solution. Extracts were centrifuged in a SpeedVac to reduce the volume. Tryptic peptides were dissolved in 20 μl of 0.1% TFA.

For LC–tandem mass spectrometry (LC-MS/MS) analysis, the peptides were separated by a gradient elution at a flow rate of 0.30 μl/min with a Thermo-Dionex UltiMate 3000 HPLC system (Thermo Fisher Scientific), coupled with a Q Exactive mass spectrometer (Thermo Fisher Scientific). The analytical column was a home-made fused silica capillary column (inner diameter, 75 μm; length, 150 mm; Upchurch, Oak Harbor, WA) packed with C-18 resin (300 Å, 5 μm; Varian, Lexington, MA). The mobile phase consisted of 0.1% formic acid, and mobile phase B consisted of 80% acetonitrile and 0.1% formic acid. The Q Exactive mass spectrometer was operated in the data-dependent acquisition mode using Xcalibur 2.1.2 software, and there was a single full-scan mass spectrum in the Orbitrap (300 to 1800 mass/charge ratio, 70,000 resolution), followed by 20 data-dependent MS/MS scans at 27% normalized collision energy [HCD (higher-energy collisional dissociation)].

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