Identification of protein vaccine candidates from Helicobacter pylori using a preparative two-dimensional electrophoretic procedure and mass spectrometry

Helicobacter pylori is an important human gastric pathogen for which the en tire genome sequence is known. This microorganism displays a uniquely compl ex pattern of binding to complex carbohydrates presented on host mucosal su rfaces and other tissues, through adhesion molecules (adhesins) on the micr obial cell surface. Adhesins and other membrane-associated proteins are imp ortant targets for vaccine development. The identification and characteriza tion of cell-surface proteins expressed by H. pylori is a prerequisite for the development of vaccines designed to interfere with bacterial colonizati on of host tissues. However, identification of membrane proteins is difficu lt using a traditional proteomics approach employing 2D-PAGE. We have used a novel approach in the identification of microbial proteins that employs a rapid preparative two-dimensional electrophoretic separation followed by:m ass spectrometry and database searches. No pre-enrichment of bacterial memb ranes was required. The entire process, from sample preparation to protein identification, can be completed in less than 18 hours, and the presence of proteins can be monitored after both the first- and second-dimensional sep arations using mass spectrometry. We were able to identify 40 proteins from a detergent-solubilized H. pylori preparation; over one-third of these wer e membrane or membrane-associated proteins. A functionally characterized lo w-abundance membrane protein, the Le(b)-binding adhesin, was found in this group. The use of this rapid 2D electrophoretic separation in proteomic stu dies of H. pylori is expected to speed up the identification of expressed v irulence proteins and vaccine targets in this and other microbial pathogens .