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Here, we present a fluorescence-based assay to measure proteolysis of the main potato protein, patatin, which was recently shown to embed emulsifying peptides. This patatin-specific assay detects protease activity with high sensitivity by measuring the increase in fluorescence emission. Emission increases due to the decreased homo-FRET (fluorescence resonance energy transfer), which results from proteolysis of extensively labelled patatin. The assay can easily be performed in a microplate format to achieve reproducible, high-throughput screening of proteolytic activity. We have validated the microplate format of the assay, which detects patatin-specific proteolytic activity efficiently by a variety of commercial proteases. By applying bottom-up/middle-down mass spectrometry-based proteomics, we have been able to map cleavage patterns for the assayed proteases. Additionally, we investigated the effect of protein labelling on proteolytic activity by comparing cleavage patterns of labelled and non-labelled patatin. We show that our approach combining high-throughput homo-FRET assays and mass spectrometry are useful to characterize specific activity and cleavage patterns of isolated proteases in vitro, which can easily be adapted for other proteases and substrates. Our vision is to adapt this assay for screening of cell cultures to facilitate characterization of e.g. membrane-bound extracellular protease activity.
|Publikationsdato||28 jan. 2020|
|Status||Udgivet - 28 jan. 2020|
|Begivenhed||Microbial Food and Feed Ingredients: Enzymes for food and feed applications - University of Copenhagen, Copenhagen, Danmark|
Varighed: 15 apr. 2020 → 17 apr. 2020
|Konference||Microbial Food and Feed Ingredients|
|Lokation||University of Copenhagen|
|Periode||15/04/2020 → 17/04/2020|
FingeraftrykDyk ned i forskningsemnerne om 'Characterizing patatin specific protease activity by high-throughput homo-FRET assay and mass spectrometry'. Sammen danner de et unikt fingeraftryk.
- 1 Igangværende
01/09/2017 → 30/12/2022
Projekter: Projekt › Forskning
- 1 Tidsskriftartikel
Emulsifier peptides derived from seaweed, methanotrophic bacteria, and potato proteins identified by quantitative proteomics and bioinformaticsYesiltas, B., Gregersen, S., Lægsgaard, L., Brinch, M. L., Olsen, T. H., Marcatili, P., Overgaard, M. T., Hansen, E. B., Jacobsen, C. & García-Moreno, P. J., 15 nov. 2021, I: Food Chemistry. 362, 12 s., 130217.
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer reviewÅben adgangFil