Ltq orbitrap elite mass spectrometer system

For Co-IP, LAC cells were infected with MPC1 overexpressing lentivirus or control lentivirus, which contained a 3 × flag sequence. Anti-flag M2 Magnetic Beads (Sigma-Aldrich, St. Louis, MO, USA) were used for detection and capture of the fusion proteins. The detailed procedures were performed according to the manufacturer’s instructions. For the liquid chromatography–MS analysis, the gel pieces with MPC1 immunoprecipitated complex or control were dehydrated in acetonitrile, dried in a speed vacuum, and digested with trypsin. The peptides were extracted from the polyacrylamide and were evaporated for MS analysis using LTQ-Orbitrap Elite Mass Spectrometer System (Thermo Scientific, Waltham, MA, USA).

786-O cells with and without HA-tagged DPF3a expression were washed with ice-cold PBS and homogenized in lysis buffer containing protease and phosphatase inhibitors. The DPF3a-associated proteins or SNIP1-associated proteins were immunoprecipitated with anti-HA beads (Thermo) or Protein G beads coupled with SNIP1 antibody for two hours. After three times washing with TBST, on-beads digestion with sequencing-grade trypsin (Promega) was carried out. Afterward, the samples were analyzed on an LTQ-Orbitrap Elite mass spectrometer system (Thermo). IP-MS data was processed by MaxQuant for label-free quantification with “match between run” function activated and database searching was against the UniProt human database.