Method for in vitro detection and monitoring of a disease by measuring disease-associated protease activity in extracellular vesicles

A method for in vitro detecting physiological enzymatic activity of a protease, wherein the protease is ADAM17, in protease-containing extracellular vesicles (EV), the method comprising:

• providing a modified peptide by combining a protease-sensitive peptide comprising 5 or more amino acids with (a) a fluorophore modification and a quencher modification, wherein the fluorophore modification is lipophilic, conferring membrane translocation potential to the peptide, or (b) an N- and/or C-terminal sequence comprising 5-20 membrane-penetrating amino acids with a fluorophore modification and a quencher modification, wherein the modified peptide comprises a protease-specific cleavage site located between the fluorophore modification and the quencher modification;
• and
• detecting physiological enzymatic activity of the protease in enriched and/or purified protease-containing EV from a plasma sample using the modified peptide, wherein the enriched and/or purified EV are EV enriched and/or purified by performing an enrichment and/or purification method comprising an antibody-based method or use of a sucrose gradient or exosome isolation reagent, and wherein said EV comprise EV that do not express CD81.