*2.8. EV Protein Extraction and Digestion*

EV pellets were resuspended in 100 μL 50 mM ammonium bicarbonate containing 0.1% Rapigest (Waters Corp., Milford, MA, USA), sonicated twice for 10 s and incubated with shaking for 1 h at room temperature. Extracts were clarified at 13,000 rpm in a microcentrifuge and protein concentration was determined using a Qubit assay kit (Thermo Fisher Scientific). Dithiothreitol was added to a final concentration of 5 mM and the sample was incubated at 65 ◦C for 30 min. Iodoacetamide was then added to a final concentration of 15 mM and the sample was incubated in the dark, at room temperature for 30 min. Sequencing grade trypsin (Promega Corp, Madison, WI, USA) was added at a 1:30 ratio and the sample was digested overnight at 37 ◦C. Trifluoroacetic acid was then added to a final concentration of 0.5% and sample was incubated at 37 ◦C for 30 min to precipitate the Rapigest. The sample was clarified at 13,000 rpm for 5 min in a microcentrifuge, dried in a vacufuge and resuspended in 20 μL 50 mM acetic acid. Peptide concentration was determined at 280 nm using a nanodrop spectrophotometer (Thermo Fisher). Five separate D4 EV preparations and three separate P1 EV preparations, all from different mHSC isolations, were individually prepared in this manner for mass spectrometry.
