Characterization of the Outer Membrane Protein Profile from Disease-Related Helicobacter pylori Isolates by Subcellular Fractionation and Nano-LC FT-ICR MS Analysis

Abstract

Because of the important role of membrane proteins in adhesion, invasion, and intracellular survival of pathogens in the host, membrane proteins are of potential interest in the search for drug targets or biomarkers. We have established a mass spectrometry-based method that allows characterization of the outer membrane protein (OMP) profile of clinical isolates from of the human gastric pathogen Helicobacter pylori. Subcellular fractionation and one-dimensional gel electrophoresis (1D-GE) analysis was combined with nano-liquid chromatography Fourier transform-ion cyclotron resonance mass spectrometry (nano-LC FT-ICR MS) and tandem mass spectrometry (MS/MS) analysis of fifteen H. pylori strains associated either with duodenal ulcers, gastric cancer, or isolated from asymptomatic H. pylori infected carriers. Over 60 unique membrane or membrane-associated proteins, including 30 of the 33 theoretically predicted OMPs, were identified from the strains. Several membrane proteins, including Omp11 and BabA, were found to be expressed by all strains. In the search for clinical markers we found that Omp26 was expressed by all disease-related strains but was only present in one out of five strains from asymptomatic carriers, which makes Omp26 a potential target for further investigation in the search for proteins unique to disease-related H. pylori strains. In addition, presence of Omp30 and absence of Omp6 seemed to be associated with H. pylori strains causing duodenal ulcer.

Differences in Genome Content among Helicobacter pylori Isolates from Patients with Gastritis, Duodenal Ulcer, or Gastric Cancer Reveal Novel Disease-Associated Genes

“…The HP0025 gene, which encodes an outer membrane protein (Omp2), was identified among the genes that were present less frequently in DU isolates. In accordance with our results, a recent report found that the protein was less frequently expressed in isolates from DU cases (10). In contrast, it is noteworthy that this gene was present in 100% of the GC isolates.…”

Recognition of the Major Cat Allergen Fel d 1 through the Cysteine-rich Domain of the Mannose Receptor Determines Its Allergenicity

“…The method for in-gel protein digestion with trypsin described by Shevchenko et al (35) was applied with some minor modifications (34). Briefly, the gel pieces were destained by washing three times in 25 mM NH 4 HCO 3 in 50% CH 3 CN and one time in 25 mM NH 4 HCO 3 in 50% CH 3 OH.…”

Proteomic analysis of highly purified prolamellar bodies reveals their significance in chloroplast development

“…The protein compositions of EPIMs (Blomqvist et al 2006) and whole etioplasts Kleffmann et al 2007) have been analysed, though no previous study has focused on the PLB proteome. We adopted a relatively new combination of analytical methods, with high potential in proteomic analysis-the one-dimensional (1-D) sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) combined with nanoliquid chromatography Fourier transform-ion cyclotron resonance mass spectrometry (nano-LC FTICR MS), an approach used with great success (Olsen et al 2004;Carlsohn et al 2006). By this method, proteins are separated by SDS-PAGE, and gel pieces from an entire sample lane are excised and subjected to mass spectrometry analysis.…”