Proteomic Analysis of Helicobacter pylori Whole Cell Proteins using the Narrow Range IPG Strips

Abstract

It has been reported that most of Helicobacter pylori proteome components appear so crowded in the region of pH 4.5~8.0 that a lot of them were inseparable in 2-DE using the broad range IPG strip. Therefore, inseparable protein spots in 2-DE profiles have to be apart from each other for improving the protein identification. Here, we attempt to examine the usability of the narrow range IPG strips for separating close spots in the broad range IPG strip at proteomic analysis of H. pylori. The whole cell proteins of H. pylori strain 26695 were separated by narrow range IPG strips (pI 3.9~5.1, 4.7~5.9, 5.5~6.7, and 6.3~8.3, respectively), followed by SDS-PAGE, and visualized by silver staining, showing that the distances between spots were widened and the total number of detectable spots was increased. Resolved protein spots were identified by the peptide fingerprinting using MALDI-TOF-MS. As a result, 87 expressed proteins were identified by the peptide fingerprinting. Of them, 23 proteins, including hydrogenase expression/formation protein, purine-binding chemotaxis protein, and ribosomal protein S6, have not been reported in the previous proteome studies of H. pylori. Thus, these results demonstrate that the high complexity proteome components could be effectively separated using the narrow range IPG strips, which might be helpful to strengthen the contents of the master protein map of the H. pylori reference strain.

Helicobacter pylori: Bacterial Strategy for Incipient Stage and Persistent Colonization in Human Gastric Niches

“…H. pylori has notoriously high chromosomal variation among isolates at the levels of both microdiversity and macrodiversity, even though proteomics display has been revealed to be nearly identical among strains. 93 , 94 The H. pylori genomic diversity includes variation in genome size, gene order, and allelic profile. About one-third of the 1600 genes predicted in the 1.6 Mbp-genome are considered to be H. pylori specific due to the absence of homologues in other organisms.…”

Proteomic Analysis of Thiol-active Proteins ofHelicobacter pylori26695

“…The supernatant containing periplasmic proteins was collected as the periplasmic fraction and was filtered with 0.2 mm nitrocellulose paper after which the pellet of osmotic shocked bacteria was resuspended in 100 ml PBS and harvested by centrifugation as the precipitant body.Two-dimensional electrophoresis (2-DE) and image analysisEluted disulfide protein was cleaned to remove contaminants with TCA precipitation and a 2-D clean up kit (Amersham Biosciences Korea). Two-DE was carried out as described previously(55). The solubilized protein sample(200 μg) was mixed with the rehydration solution containing 8 M urea, 4% CHAPS, 10 mM DTT, and 0.2% carrier ampholytes (pH 5.0~8.0 and 6.0~11.0) to a final volume of 300 μl, and was applied to immobilized pH gradient (IPG) strips (17 cm; Bio-Rad, Hercules, CA, USA) of pH 5.0~8.0 and 6.0~11.0 in a reswelling tray (Bio-Rad).…”

“…Spot intensities of each sample were documented and analyzed using the PDQUEST 2-D Gel Analysis Software Version 7 (Bio-Rad) installed on a Magic Station M5660 (Samsung, Korea).Destaining and in-gel digestion of protein spotsThe silver-stained spots were excised from the 2-DE gels and were transferred into microcentrifuge tubes. The spots were de-stained with fresh chemical reducers in a 1:1 ratio of 30 mM potassium ferricyanide and 100 mM sodium thiosulfate, as described previously(55,56), with occasional mixing until the brownish color disappeared.The gel pieces were rinsed three times with distilled water to stop the reaction. Ammonium bicarbonate (500 μl of 200 mM) was added to cover the gels for 20 min, and was then discarded.…”