Plasma Extracellular Vesicle Fractionation

The whole procedure was performed at 4 °C. There were four tubes of pooled plasma from four groups each containing around 5 mL of plasma specimens. The samples were subjected to sequential centrifugation and ultracentrifugation to fractionate extracellular vesicles using a modified protocol as described previously [18 (link),19 (link)]. Briefly, sonicated plasma (5 ×1 min) was centrifuged at 4000× g twice for 30 min and then at 12,000× g for 30 min to collect the pellets (4000× g—P1 and 12,000× g—P2). The resulting supernatant was diluted five times with 1X PBS and ultra-centrifugated (swinging bucket rotor: SW 55 Ti, k-factor = 48) at 30,000× g for 2 h to collect the pellet (P3). The supernatant was used for the isolation of the SEV-rich (exosome) fraction as reported earlier [18 (link)]. P3 was washed with 1X PBS twice and lyophilized. P2 and P3 were dissolved using 50 µL of ice-cold dissolution buffer (6% sodium dodecyl sulfate; 20 mM dithiothreitol, 100 mM tris-HCl with Complete Protease Inhibitor Cocktail (COMPLETE, (Roche; Mannheim, Germany)), pH 7.75) by brief vertexing. Protein quantization was performed using the 2-D Quant Kit (Amersham Biosciences, Piscataway, NJ, USA). P2 and P3 were combined to obtain MEVs for proteomics sample preparation.