Identification of Protein Vaccine Candidates from Helicobacter pylori Using a Preparative Two-Dimensional Electrophoretic Procedure and Mass Spectrometry

Abstract

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.

A Phospholipase D-dependent Process Forms Lipid Droplets Containing Caveolin, Adipocyte Differentiation-related Protein, and Vimentin in a Cell-free System

“…If the spectra were not good enough, the samples were cleaned up and concentrated with a ZipTip containing C 18 material (Millipore) before the MS run. When the MALDI-TOF analysis was inconclusive, tandem MS/MS was performed with the quadrupole time-of-flight technique as described (49), and proteins were identified with Mascot software.…”

Identification of Surface Proteins of Helicobacter pylori by Selective Biotinylation, Affinity Purification, and Two-dimensional Gel Electrophoresis

“…In H. pylori, these techniques yielded enriched outer membrane fractions, and many putative surface proteins have been found. However, these preparations are heavily contaminated by inner membrane components (13,14), so that independent evidence, such as specific antibody binding to intact bacteria, is necessary to verify true surface proteins (15). Five outer membrane proteins with the typical ␤-barrel structure were identified by their anomalous temperature-dependent migration in polyacrylamide gels (14), but surface proteins with different structures escaped this method.…”

Two Dimensional Gel Electrophoresis in Cancer Proteomics

“…Techniques such as differential extraction , subcellular fractionation (Taylor et al 2000, Morel et al 2000, chromatography , or prefocusing in a preparative IEF device such as the Rotofor® system (Masuoka et al 1998, Nilsson et al 2000 have been used to reduce the complexity of samples.…”