Surface proteins of SEC purified EVs of A. perfoliata were removed through hydrolysis with trypsin as previously described [49 (link)]. Briefly, SEC purified EVs were diluted with PBS to a final concentration of 200 μg in 250 μL total volume. Sequencing grade modified trypsin (100 μg/mL; Roche, U.K) was added to the EVs obtained a final concentration of 50 μg/mL and incubated for 5 min at 37 °C. The treated EVs were then centrifuged for 1 h at 100,000× g at 4 °C. The resulting supernatant was stored at −20 °C prior to gel free mass spectrometry analysis.
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