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 entire genome sequence is known. This microorganism displays a uniquely complex pattern of binding to complex carbohydrates presented on host mucosal surfaces and other tissues, through adhesion molecules (adhesins) on the microbial cell surface. Adhesins and other membrane-associated proteins are important targets for vaccine development. The identification and characterization of cell-surface proteins expressed by H. pylori is a prerequisite for the development of vaccines designed to interfere with bacterial colonization of host tissues. However, identification of membrane proteins is difficult 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 mass spectrometry and database searches. No pre-enrichment of bacterial membranes 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 separations using mass spectrometry. We were able to identify 40 proteins from a detergent-solubilized H. pylori preparation; over one-third of these were membrane or membrane-associated proteins. A functionally characterized low-abundance membrane protein, the Leb-binding adhesin, was found in this group. The use of this rapid 2D electrophoretic separation in proteomic studies of H. pylori is expected to speed up the identification of expressed virulence proteins and vaccine targets in this and other microbial pathogens.
Nilsson
C L
CL
Institute of Medical Biochemistry, Göteborg University, Sweden. carol.nilsson@medkem.gu.se
Larsson
T
T
Gustafsson
E
E
Karlsson
K A
KA
Davidsson
P
P
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis*; D001428:Bacterial Vaccines / Q000032:analysis*; D016208:Databases, Factual; D004591:Electrophoresis, Polyacrylamide Gel; D016480:Helicobacter pylori / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019151:Peptide Library
D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D019151:Peptide Library