LC-MS/MS analysis of the modified active site peptide of BioZ C115S

These analyses were performed by the Protein Sciences Facility of the Roy J. Carver Biotechnology Center, University of Illinois. The BioZ C115S mutant protein was digested with Glu-C and trypsin in 50 mM ammonium acetate. Liquid chromatography-mass spectrometry analysis was perform using a Dionex Ultimate 3000 RSLC nanoflow UPLC system (Thermo Scientific) coupled to a Fusion Tribrid Orbitrap mass spectrometer (Thermo Scientific). An Acclaim PepMap RSLC column (75 µm × 15 cm) was used for the separation using a 60 min gradient (1–60%) of acetonitrile in 0.1% formic acid. The spectra were obtained using the Universal method utilizing data-dependent acquisition and maximum parallelizable throughput. The collected spectra were processed using Mascot Distiller and analyzed using MASCOT run against a customized database containing the sequence of the protein of interest.