Ltq orbitrap xl hybrid ion trap mass spectrometer

We grew the E. coli MC4100relA⁺ strain or its derivative MC4100relA⁺ ΔclpP strain carrying or not carrying plasmid pzwf in LB medium with or without ampicillin at 37°C, with shaking at 220 rpm, overnight. We diluted these cells 1:100 in LB medium with ampicillin and grew them at 37°C, with shaking at 220 rpm, to mid-logarithmic phase (OD₆₀₀ of 0.6). After inducing these cells by incubating them with 1 mM IPTG at 37°C for 30 min, we collected them by centrifugation at 14,000 rpm for 5 min and analyzed them by SDS-PAGE. We excised the Zwf band from the gel, digested it with trypsin, and then analyzed it by liquid chromatography-mass spectrometry (LC-MS) using an LTQ Orbitrap XL Hybrid Ion Trap mass spectrometer (Thermo Scientific).

For the enrichment of the antigen against the XC246 autoantibody, a SNU638 cell lysate prepared with RIPA cell lysis buffer was immunoprecipitated using XC246 antibody-conjugated beads as described above. Subsequently, the beads were treated with 0.1 M glycine buffer (pH 2.5) to elute the target antigen, and the eluate was neutralized with 1.0 M Tris buffer (pH 8.0). The eluates were then separated using 10% SDS-PAGE, followed by western blotting or Coomassie brilliant blue staining at 25°C for 2 h. The Coomassie-stained band corresponding to the XC246 antigen band was confirmed using western blotting. The XC246 antigen band was then excised and in-gel digested with trypsin (Promega Corporation). Protein identification was performed via ESI-TRAP mass spectrometry (LTQ Orbitrap XL Hybrid Ion Trap Mass Spectrometer; Thermo Fisher Scientific, Inc.) and Mascot database search at the Korea Basic Science Institute (Ochang, Korea).