Protein crosslinking and LC–MS/MS analysis

Equal amount (w/w) of BS3 (bis[sulfosuccinimidyl] suberate) was added to the protein mixture, which was incubated at room temperature for 1 h. The reaction was terminated by adding 0.5 M ammonium bicarbonate to a final concentration of 20 mM for 10 min incubation. The SDS-PAGE was used to separate the crosslinked protein and stained with Coomassie Blue G-250. The gel bands of interest were cut into pieces. Sample was digested by trypsin with prior reduction and alkylation in 50 mM ammonium bicarbonate at 37 °C overnight. The digested products were extracted twice with 1% formic acid in 50% acetonitrile aqueous solution and dried to reduce volume by speedvac.

For LC–MS/MS analysis, the peptides were separated by a 65 min gradient elution at a flow rate 0.300 µl/min with the Thermo EASY-nLC1200 integrated nano-HPLC system which is directly interfaced with the Thermo Q Exactive HF-X mass spectrometer. The analytical column was a home-made fused silica capillary column (75 µm ID, 150 mm length; Upchurch, Oak Harbor, WA) packed with C-18 resin (300 A, 3 µm, Varian, Lexington, MA). Mobile phase A consisted of 0.1% formic acid, and mobile phase B consisted of 100% acetonitrile and 0.1% formic acid. The mass spectrometer was operated in the data-dependent acquisition mode using the Xcalibur 4.1 software and there is a single full-scan mass spectrum in the Orbitrap (300–1800 m/z, 60,000 resolution) followed by 20 data-dependent MS/MS scans at 30% normalized collision energy. Each mass spectrum was analyzed using the Thermo Xcalibur Qual Browser and pLink 2⁶⁹ for the database searching and cross-linking analysis.