Maxent 1

The tested samples, consisting of 2 µM of CTSB with 500 µM of 3-BrPA in 50 mM of acetate buffer (with 1 mM of EDTA; pH 5.0) and control samples (2 µM of CTSB in 50 mM of acetate buffer (with 1 mM of EDTA; pH 5.0)) were incubated for 10 min at 37°C prior to separation. Mass spectrometric studies were conducted using a Nano-Acquity UPLC Q-TOF/MS (Waters, Milford, USA) system. The chromatograph was equipped with an Acquity UPLC HSS C18 analytical column (1 mm × 100 mm; 1.8 μm) with the flow-rate maintained at 50 μL/min and the injection volume at 3 μL. The separation was held for 15 min at 35°C. The mobile phase constituents were solvent A -0.1% formic acid in water -and solvent B, 0.1% formic acid in acetonitrile. The elution took place in a gradient system: 0-1 min: 15% B; 2 min: 25% B; 8 min: 60% B; 10 min: 85% B; 11 min: 85% B; 11.5 min: 15% B; 15 min: 15% B. The eluted CTSB was analyzed using Xevo G2-Q-TOF (Waters) mass spectrometer with an electrospray ionization source in positive ionization mode (ESI+). The capillary voltage was set at 3.0 kV, and the cone voltage for CTSB was set at 40 V. The cone gas flow was maintained at 80 L/h, and the source temperature was set at 100°C. Leucine Enkephalin (Waters) was used as the lock mass solution. Data was collected from m/z 200 to m/z 2000. The mass spectra were deconvoluted with MaxEnt 1 (Waters).