A homo-FRET assay for patatin-specific proteolytic activity

The potato protein patatin embeds bioactive peptides that require targeted hydrolysis to be released as promising food additives. This study presents a patatin-specific protease assay for assessing a wide range of protease activities in high-throughput format. Conjugating patatin to the amine reactive fluorogenic BODIPY FL dye provided a stable protease substrate with efficient homo-FRET quenching at a low degree (7-8) of labeling. Compared to commercial BODIPY-casein, BODIPY-patatin provided higher fluorescence enhancement (by de-quenching) at high protease concentrations, while the sensitivity was generally comparable for both highly specific (e.g. Trypsin) and industrial relevant proteases (e.g. Alcalase and Neutrase) at low doses. For Chymotrypsin, BODIPY-patatin provided a 39 % response improvement at 5 ng dose. A peptide-centric analysis of mass spectrometry-based bottom-up proteomics data identified several BODIPY-labeling sites with varying occupancies in patatin, indicating heterogenous labeling under the applied conjugation conditions. BODIPY-labeled patatin complements commercial BODIPY-labeled casein as a globular, plant-based alternative for screening of proteolytic activity.