Proteinase K-based Extracellular Vesicle Protein Extraction

The EVs derived from calli were treated with Proteinase K (20 µg/mL) for 1 h at 37 °C, with intermittent gentle mixing every 10 min to ensure uniform exposure of the vesicles to the enzyme. Following Proteinase K treatment, the proteolytic activity was curtailed by introducing 5% PMSF into the samples. The subsequent inhibition process involved incubation for 10 min at room temperature. Further inactivation of the proteinase was achieved by subjecting the samples to a heat treatment at 90 °C for 5 min. The extraction of total proteins followed established protocols, as previously described [77 (link)]. Protein extracts obtained from the EV samples were separated by 12.5% SDS-PAGE and visualized by Coomassie staining. Upon completion of electrophoresis, gel slices containing the protein components of interest were meticulously excised into smaller fragments. The excised gel slices were subjected to in-gel digestion with trypsin (Trypsin V511; Promega, Madison, WI, USA) using a previously described procedure [83 (link)]. Following trypsin digestion, the resulting peptide mixture was dried using a Concentrator plus (Eppendorf, Hamburg, Germany).