Proteomics Analysis of H. pylori

H. pylori cell pellets were lysed and its protein was extracted using Norgen's Proteospin™ total protein purification kit (Norgen Biotek Corporation, Canada). Cells were resuspended in 50 μl lysis buffer and centrifuged at 14,000 × g for 2 min. The supernatant was transferred into a filter column fitted in an elution tube, and centrifuged at 14,000 × g for 1 min. One microliter of protease inhibitor (Halt Protease and Phosphatase Inhibitor; Thermo Fisher Scientific) was added to the tube. Protein concentrations were determined by Bradford assay (Bio-Rad, USA). Each tube of protein sample was reduced with a volume of 10 mM dithiothreitol (DTT; Bio-Rad), and alkylated in the dark with a volume of 20 mM iodoacetamide (IAA; Bio-Rad). The samples were incubated at room temperature for 30 min and added with Pierce™ trypsin protease (Thermo Scientific) to digest the proteins at 1:50 trypsin:protein. The samples were then incubated at 4°C for 30 min, and subsequently at 37°C for overnight. The extracted protein was then treated for liquid chromatography mass spectrophotometry according to a previous study (Chan et al., 2015 (link)).