K factor 280

EV enrichment was performed from 1 mL plasma with double centrifugation at 100,000 × g, 1 h, 4 °C using an Avanti J-30i centrifuge with a J A-30.50 fixed angle rotor, k-factor 280 (Beckman Coulter, Brea, CA, USA). After initial centrifugation, EVs were washed in 1 mL 0.22 µm filtered phosphate-buffered saline (PBS). The EV enriched pellet was resuspended in 20 µL filtered PBS for mass spectrometry and in 100 µL for EV characterisation.

EV isolation was performed from 1 mL plasma with one centrifugation at 20,000×g for 30 min at 4 °C using an Avanti J-30i centrifuge with a J A-30.50 fixed-angle rotor with a k-factor 280 (Beckman Coulter, Brea, CA, USA). The supernatant from the initial spin of the 20 K pellet was used to prepare the 100 K pellet (100,000×g for 1 h at 4 °C). Succeeding the initial centrifugation step for each pellet preparation, the resultant EVs were washed in 1 mL phosphate-buffered saline filtered by a 0.22 µm filter. The final enriched 20 K (microvesicles; large EVs) and 100 K (exosomes; small EVs) samples were resuspended in 20 µL filtered phosphate-buffered saline prior to MS analysis. The samples were lysed and solubilized in 5% sodium dodecyl sulfate containing 50 mM triethylammonium bicarbonate, pH 7.55. Alkylation and tryptic digestion were performed using S-TrapTM Micro Spin Columns (Protifi, NY, USA) essentially as previously described [16 ]. Proteins were cleaved using PierceTM Trypsin protease, MS Grade (Thermo Fisher Scientific, Waltham, MA, USA) and peptide concentrations were measured by fluorescence using an EnSpire microplate reader (Perkin Elmer, Waltham, MA, USA). Samples were resuspended in 0.1% formic acid and injected with an amount of 1 µg in case of 20 K sample and 0.75 µg in case of 100 K sample.