Mass Spectrometry-based Protein Identification

Protein digestion and MS analysis were executed as previously described^{47 (link)}. In brief, proteins were digested with trypsin, vacuum-freeze-dried, and resuspended in anhydrous acetonitrile solution, then desalted with MonoTIPTM C18 Pipette Tip (GL Sciences, Tokyo, Japan). Peptide samples were analyzed with an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Then raw data were analyzed using Proteome Discoverer (Thermo Fisher Scientific) and Spectronaut (Omicsolution Co., Ltd., Shanghai, China) software. Protein and peptide FDRs were set to 1%. For Co-IP, cell lysates were incubated with IgG (Santa Cruz Biotechnology) and protein A/G Sepharose beads (Santa Cruz Biotechnology) at 4 °C for 1 h. The supernatant was mixed with the appropriate primary antibody overnight at 4 °C prior to a 4 h incubation with protein A/G Sepharose beads. After washing with PBS and lysis buffer, the beads were mixed with 5× SDS/PAGE loading buffer for Western blot analysis.