Peptide Characterization by LC-MS/MS

Peptides were dissolved in Optima^® LC/MS water (Fisher Chemical, Waltham, MA, USA) at a concentration of 1 mg/mL. Peptide solutions were infused at a flow rate of 10 µL min⁻¹ through the electrospray ionization source of a LCMS IT-TOF mass spectrometer (Shimadzu Scientific Instruments, Columbia, MD, USA). The CDL and heat block were set at 200 °C. Nebulization gas was set to 1.0 L min⁻¹, and drying gas was set to “on”. Accurate mass spectra were collected in the positive ionization mode from m/z 200 to 2000. Tandem mass spectra were acquired using ultra-pure argon (Airgas, Alexandra, VA, USA) as the collision gas at an arbitrary collision energy setting of 40% and an arbitrary flow rate of 50%. The precursor ion accumulation time in the ion trap was set at 10 ms. Raw data files were processed in LCMSSolution software version 3.7 (Shimadzu Scientific Instruments, Columbia, MD, USA) and, when necessary, converted to the mzXML format using the LCMS Solution convert tool. Peak picking and theoretical product ion m/z generation were performed in mMass v5.5 [41 (link)].