Mass spectrometry-based proteomic strategies applied to Helicobacter pylori. A Search for candidate vaccine antigens

Abstract

This thesis describes the development and refinement of proteomic strategies and the application of these on the human gastric pathogen H. pylori. The purpose was to identify proteins, and in particular membrane proteins, as potential targets for vaccine development. A semi-preparative method based on liquid-phase separation of whole cell extracts was found to be useful. However, in order to perform a more comprehensive analysis of the outer membrane protein profile, and to identify proteins specific for disease-related H. pylori strains, a new strategy combining subcellular fractionation with high-sensitivity nano-liquid chromatography Fourier transform-ion cyclotron resonance mass spectrometry (nano-LC FT-ICR MS) analysis was used. Differences among outer membrane proteins (OMPs) were compared in clinical bacterial isolates of asymptomatic carriers and of patients with duodenal ulcer or gastric cancer. Several potential targets for a vaccine were identified and proteins that may be used as diagnostic markers or should be subjected to more detailed studies were identified. These results document the importance of using nano-LC coupled to the powerful FT-ICR MS method for high confidence identification of low-abundance components of a complex mixture. The protein expression patterns of the mouse-adapted H. pylori strain SS1 and its isogenic HpaA mutant strain were examined using the two strategies. Whole cell extracts were analyzed by traditional 2D- gel electrophoresis (2D-GE) and differential in-gel electrophoresis (DIGE) technology, and the OMP profiles were compared using the fractionation and nano-LC MS approach. Only minor differences in the protein expression pattern were found between the two strains. HpaA was identified in the SS1 strain, but could not be detected in the mutant strain, although the low-abundance OMP BabA was readily identified. Infection studies in mice demonstrated that HpaA is essential for H. pylori colonization, and this protein is therefore a candidate vaccine antigen. In order to evaluate the potential of using mass spectrometry to localize surface-exposed peptides of a protein (potential immunogenic epitopes), we performed chemical cross-linking studies of the urease complex of H. pylori. This protein is easy to prepare and the known three-dimensional structure facilitates the interpretation of results by molecular modeling. Although cross-linked peptides were identified, the conclusion is that this potentially helpful technique requires additional developmental efforts to be practically useful.