The proteins identified from Helicobacter pylori strain TN2wt-derived extracellular vesicle (HpEV-TN2wt)

Helicobacter pylori strain TN2wt was suspended in Brucella broth supplemented with 5% depleted exosomes FCS. The initial optical density at 600 nm of the bacterial suspension was adjusted to 0.02 in 200 mL of Brucella broth + 0.5% depleted exosome FCS. H. pylori was grown in a 500 mL flask under microaerophilic conditions, shaking at 100 rpm, at 37oC for 48 h. The H. pylori cells were removed from the culture supernatant by centrifugation at 15,000 x g, for 15 min at 4oC and the bacteria-free supernatant was then filtered through a 0.45 m filter. HpEVs in culture supernatant of H. pylori were harvested by ultracentrifugation at 200,000 x g for 90 min at 4oC using Himac CP80WX Preparative ultracentrifuge (HITACHI, Tokyo, Japan). After removing the supernatant, the HpEVs were washed in 20 mL of 0.9% NaCl solution and collected by ultracentrifugation at 200,000 x g for 90 min at 4oC. HpEVs were resuspended in saline and stored at -80oC. The HpEVs were lysed with 2% sodium dodecyl sulfate/ 7M urea, and equal amounts of proteins from each sample were precipitated with acetone. The proteins were then denatured and reduced with 50% trifluoroethanol and 4 mM dithiothreitol followed by alkylation of the free cysteine residues and trypsinization. The desalted peptides were then separated using liquid chromatography. The mass spectrometer was operated in information-dependent acquisition mode, and acquired spectra were searched against the H. pylori TN2wt protein database.