Human gastric adenocarcinoma AGS cells were infected with H. pylori Tx30a or TN2wt at a multiplicity of infection = 100 and incubated at 5% CO2,37oC for 48 h. The cells/debris were removed from the cell culture supernatant by centrifugation at 2,000 x g for 30 min at 4oC and filtered through a 0.45 m filter. The EVs were collected by ultracentrifugation at 110,000 x g for 70 min using Himac CP80NX Preparative Ultracentrifuge (HITACHI, Tokyo, Japan). After removing the supernatant, the EVs were washed with PBS and then collected by ultracentrifugation at 100,000 x g for 70 min at 4oC. Finally, the EVs were resuspended in PBS, and stored at -80oC. The AGS-derived EVs 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 AGS cell protein database. Differential protein expression was analyzed to determine the relative abundances of proteins in EVs derived from AGS cells infected with H. pylori Tx30a or TN2wt, compared to non-infected cells. H. pylori proteins in AGS-derived EV, the spectra were searched against the H. pylori ATCC 51932 (Tx30a) and TN2wt protein database.