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/).