Western blot analysis
Total protein concentration of EVs was determined with the Bradford Protein Assay Kit (ThermoFisher) [24]. Samples were lysed in reducing conditions with β-mercaptoethanol and 30µl of EVs were loaded per lane. EVs were analyzed by Western Blot with primary antibodys anti-CD9 (Invitrogen), anti-CD63 (Invitrogen), anti-Syntenin-1 (Novus Biologicals), anti-TSG101 (Abcam), anti-S100A9 (Cell signaling technology), anti-CDC42 (Cell signaling technology) and anti-Ficolin-2 (Abcam). Goat anti-rabbit (GAR) or rabbit anti-mouse (RAM) HRP-conjugated secondary antibodies were used (Jackson Laboratory, Bar Harbor, Me).
Proteins digestion and peptides fractionation
For proteins identification and relative quantification by label free approach, 10 paired samples of human EVs (AnEVs versus BEVs) were used, each constituted by pools of 2 individual samples. The same amount of total protein from each pool was tryptic digested and the resulting peptides were pre-fractionated in 7 fractions by High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher Scientific).
Protein quantification by liquid chromatography and mass spectrometry in tandem
Peptides fractions were analyzed by reversed phase liquid chromatography and mass spectrometry in tandem (RP-LC-MS/MS) in an EASY‑nLC 1000 System coupled to a Q-Exactive HF mass spectrometer through a Nano-Easy spray source (Thermo Scientific, Bremen, Germany). All data were acquired in data-dependent acquisition (DDA) mode with Xcalibur 4.0 software. Peptide identification was carried out using Mascot v. 2.6.1 search engine through Protein Discoverer 2.3 Software (Thermo Scientific). Protein quantitation was performed by spectral counting with Proline 2.0 software (PL).
System biology Analysis
We used Ingenuity pathway analysis (IPA) software (Qiagen) to interpret the differentially expressed data including biological processes, canonical pathways and gene networks. Gene list contained in the main canonical pathways observed by IPA were analyzed in STRING (https://string-db.org/) and functional protein associated networks were visualized. The principal component analysis (PCA) and the heat map were realized in the website ClustVis (https://biit.cs.ut.ee/clustvis/).