Proteomic analysis of the sarcosine-insoluble outer membrane fraction of Helicobacter pylori strain 26695

Abstract

Helicobacter pylori causes gastroduodenal disease, which is mediated in part by its outer membrane proteins (OMPs). To identify OMPs of H. pylori strain 26695, we performed a proteomic analysis. A sarcosine-insoluble outer membrane fraction was resolved by two-dimensional electrophoresis with immobilized pH gradient strips. Most of the protein spots, with molecular masses of 10 to 100 kDa, were visible on the gel in the alkaline pI regions (6.0 to 10.0). The proteome of the OMPs was analyzed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Of the 80 protein spots processed, 62 spots were identified; they represented 35 genes, including 16 kinds of OMP. Moreover, we identified 9 immunoreactive proteins by immunoblot analysis. This study contributes to the characterization of the H. pylori strain 26695 proteome and may help to further elucidate the biological function of H. pylori OMPs and the pathogenesis of H. pylori infection.

FIG. 1.

Sarcosine-insoluble fraction of H. pylori 26695 were separated by 2-DE with an IPG strip, followed by SDS-12.5% PAGE. Spots were detected by silver staining. The circled proteins were identified by MALDI-TOF-MS. (A) IPG strip, pH 3.0 to 10.0; (B) IPG strip, pH 6.0 to 11.0. Strain 26695 was grown as described in Materials and Methods, and 300 μg of sarcosine-insoluble fraction was loaded in the first dimension. Identified proteins are indicated by spot numbers in Table 1. Molecular size markers are shown on the left in kilodaltons.

FIG. 2.

Comparison of gel-estimated and calculated molecular weights and pI values of the protein spots of H. pylori 26695. (A) Molecular masses; (B) pI values. The theoretical values were referred from the NCBI database of H. pylori strain 26695.

FIG. 3.

2-DE immunoblot with the sarcosine-insoluble fraction of H. pylori resolved by IEF in the pH 6 to 11 and SDS-12.5% PAGE gel. The membrane was probed with a pool of 300 sera from seropositive patients (dilution 1:10). Numbers correspond to the identified proteins in Table 1. Molecular size markers are shown on the left.