Proteomic analysis
Mass spectrometry proteomic analysis was conducted on LEAP‐purified pEVs to identify the proteins present. A modified Folch extraction was performed on the samples to separate peptides and other water‐soluble components from the lipid solution. The aqueous solution was then injected into the column for analysis on a U3000 HPLC in‐line (Thermo Fisher, San Jose, CA, USA) with an Orbitrap Lumos Fusion (Mass Spectrometry and Proteomics Facility, Bio21 Institute, Australia). The data from each analysis were searched using the Protein Discoverer 2.4 software platform (Thermo Fisher, San Jose, CA, USA). The search parameters were 5 ppm tolerance for the precursor ion and 0.02 Da tolerance for the product ion. Enzymatic cleavage with trypsin with two allowable missed cleavages. Fixed modification was Carbamidomethylation on Cys; variable modifications were oxidation on Met and deamidation on Asn & Gln. The search database was Swissport, all species. The search results were then manually curated.