The quantitative proteomic analysis of Pseudomonas aeruginosa treated with or without Staphylococcus aureus-derived extracellular vesicles (SaEVs)

Pseudomonas aeruginosa was prepared and adjusted to an initial bacterial number of OD600 = 1.0 in Tryptic soy broth with or without 5 μg/mL SaEVs. After incubation at 37°C for 24 h, the bacterial cells were collected by centrifugation at 1,100 x g, 4°C for 15 min, followed by washing twice with phosphate-buffered saline (PBS). The bacterial cells were lysed with 2% sodium dodecyl sulfate/ 7M urea, and equal amounts of protein from each sample were precipitated with acetone. The proteins were denatured with 50% trifluoroethanol and reduced using 4 mM dithiothreitol. Free cysteine residues were alkylated prior to trypsinization, and the desalted peptides were then separated using liquid chromatography. The mass spectrometer was operated in information-dependent acquisition mode. Acquired spectra were searched against the proteins of P. aeruginosa strain ATCC 15692. Differential protein expression was analyzed to determine the relative abundances of proteins in SaEV-treated P. aeruginosa compared to non-treated P. aeruginosa.