J Bacteriol Virol.  2007 Dec;37(4):203-212. 10.4167/jbv.2007.37.4.203.

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

• Park JW
• Lee SG
• Song JY
• Jun JS
• Joo JS
• Youn HS
• Seo JH
• Kang HL
• Baik SC
• Lee WK
• Cho MJ
• Rhee KH

• KMID: 1513579
• DOI: http://doi.org/10.4167/jbv.2007.37.4.203

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.

Keyword

Narrow range IPG strip; Helicobacter pylori; Proteomics

MeSH Terms

Chemotaxis
Electrophoresis, Polyacrylamide Gel
Helicobacter pylori*
Helicobacter*
Hydrogen-Ion Concentration
Hydrogenase
Peptide Mapping
Proteome
Proteomics
Ribosomal Protein S6
Silver Staining
Hydrogenase
Proteome
Ribosomal Protein S6

Figure

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  Figure 1. Two-dimensional-gel electrophoresis of whole cell proteins from H. pylori strain 26695. Whole cell proteins solution was separated on a narrow range IPG strips, followed by 12.5% SDS-PAGE, and visualized by silver staining. The original gel size was 20×18×0.15 cm. (A) pH 3.9∼5.1; (B) pH 4.7∼5.9; (C) pH 5.5∼6.7; (D) pH 6.3∼8.3.

• 

  Figure 2. The composite profile of the whole cell proteins of H. pylori strain 26695 resolved in the 2-DE using the narrow pH range IPG strips. The whole cell proteins of H. pylori strain 26695 was subjected to 2-DE using IPG strips of 3.9∼5.1, 4.7∼5.9, 5.5∼6.7, and 6.3∼8.3 ranges, the gels were visualized with silver staining. The 4 kinds of images were taken up and analyzed with PDQUEST software, and excluded with the overlapping zones of each right and left termini, and then combined by PHOTOSHOP software to generate the composite one ranged from pI 3.9 to pI 8.3. The marked protein spots have been identified and listed in Table 1.

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  Figure 3. Blow-up images of two different regions in 2-DE gels of H. pylori whole cell proteins resolved by broad (pH 3.0∼10.0), intermediate (pH 5.0∼8.0), narrow (pH 4.7∼5.9) pH strips. Here, the narrower is the pH range of the strip, the higher resolution does the spots profile show in which the resolution and the number of spots are improved. HtpG spot appears in the gel of narrow pH strips but did not in otherwise.

Reference

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