Proteomic analysis of extracellular membrane vesicles from Pseudomonas aeruginosa (PaOMVs)

PaOMVs in the culture supernatant of P. aeruginosa were harvested by ultracentrifugation at 100,000 × g, 4℃ for 90 min using a Himac CP80WX Preparative Ultracentrifuge (HITACHI, Tokyo, Japan). After removal of the supernatant, the pellet was washed twice and suspended in ice-cold sterile phosphate-buffered saline (PBS). PaOMVs were then isolated from other damaged membranes, aggregated proteins and non-membranous proteins by step-gradient ultracentrifugation using OptiPrep™ Density Medium (Sigma Aldrich, St. Louis, MO). After centrifugation at 100,000 × g, 4℃ for 16 h, the substances in all six fractions (F1-F6) were collected, diluted in PBS, and harvested by ultracentrifugation at 100,000 × g, 4℃ for 3 h. The pellet from each fraction was washed twice and resuspended in a suitable volume of ice-cold sterile PBS.