Human neutrophil elastase

The diluent for all the tested compounds, protein and substrate (except KLK7, which was done with 10 mM Tris pH 7.8) was 1xPBS pH 8.0–8.9. The serially diluted ligands were mixed with diluted bovine trypsin (Sigma, Dorset, U.K.) or activated KLK14, or KLK5, or activated KLK8 or matriptase (catalytic domain only, >90% purity by SDS-PAGE, endotoxin <0.1 ng/μg, R&D, Minneapolis, U.S.) or activated KLK7 or human neutrophil elastase (purified from human plasma, >95% purity by SDS-PAGE, Abcam, Cambridge, U.K.) or porcine pancreatic elastase 1 (Sigma, Dorset, U.K.) or human plasma kallikrein or human plasma plasmin (purified from human plasma, ≥95% purity by SDS-PAGE, BioVision, Milpitas, U.S.). 20μL of the ligand-protein mixture was pipetted to a well of a 96-well microplate in triplicates. The reaction was started by addition of 90–100 μL of the diluted substrate solution (contained 0.05% v/v Brij35) and the plate incubated at 25°C but 37°C for KLK7. RFU reading for each well was taken 6 minutes (trypsin) or 15 minutes (KLK14, KLK5, KLK8, matriptase, human neutrophil elastase, porcine pancreatic elastase 1, plasma kallikrein and plasmin) or 20 hours (KLK7) after substrate addition.

Pro-D-BMAP18 (500 ng) was mixed with Human Neutrophil Elastase (Human Neutrophil Elastase, Abcam, Cambridge, UK) at a molar ratio of 100:1, respectively, and incubated at 37 °C in 50 μL HEPES 100 mM pH 7.0. Then, 15 μL of the mixture were withdrawn at different time points, the reaction was stopped by dilution in 15 μL of ice-cold H₂O + 0.1% (v/v) TFA and frozen at −20 °C until HPLC-MS analysis. Each sample was analysed by HPLC using the analytic column Symmetry^® C18 (100 Å, 3 μm, 3 mm × 100 mm; Waters, Billerica, MA, USA). The elution was performed by a linear gradient of solvent B (0.05% v/v TFA in AcCN) in A (0.05% v/v TFA in water) from the 20% (v/v) to the 35% (v/v) of B in 15′ under a flow rate of about 15 μL/min. The HPLC was connected to the mass analyser HCT ultra (Bruker Daltonics, Billerica, MA, USA) equipped with an electrospray ionization (ESI-MS) system and using a capillary voltage of 200 V.