Fig. 2.

Experimental workflow to identify and validate host proteins that are significantly altered by H. pylori infection and which are associated with gastric carcinogenesis in humans. Mongolian gerbils were challenged with Brucella broth (negative control, n = 3) or with carcinogenic H. pylori strain 7.13 (n = 3). Two biological replicate experiments were performed. Gerbil gastric tissue was harvested 6 weeks post-challenge to assess H. pylori colonization, inflammation, and global gastric proteomic changes in vivo. Proteins from pooled lysates from gastric tissue were digested, peptides were labeled with iTRAQ reagents, and quantitative analyses were performed following 2D LC-MS/MS on a Q Exactive Plus mass spectrometer. Data were searched and proteins were quantified using Spectrum Mill. Quantitative proteomic analysis from two biological replicate experiments quantified a total of 2764 proteins, 166 of which were significantly altered in abundance following H. pylori infection, compared with uninfected samples. Ingenuity Pathway Analysis (IPA) was used to identify biologically relevant canonical signaling pathways and disease pathways significantly altered by H. pylori. Protein targets were selected and validated in in vitro human gastric epithelial cells and ex vivo primary human gastric monolayers by Western blot analysis. Further, these targets were validated in in vivo gerbil gastric tissue and human gastric tissue specimens from patients at high risk for gastric cancer by immunohistochemistry.