Application of Hemin-Agarose Affinity Chromatography to Enrich Proteome Components of Helicobacter pylori Strain 26695

The whole cell extract of Helicobacter pylori strain 26695 was treated with the hemin-agarose resin and the bound fraction was analyzed by 2-Dimensional electrophoresis. The 2-D-PAGE-displayed spots were eluted and analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Among the 120 spots processed, 94 protein spots were identified to represent 58 genes. Forty-five protein spots that represented thirty-four genes were newly identified in this study, including iron-containing proteins and hemin-containg proteins such as fumarate reductase, iron-sufur subunit(FrdB), ribonucleoside diphosphate reductase, beta subunit (NrdB), glutamyl-tRNA reductase (HemA), nikel-cobalt-cadnium resistance protein (NccB), and porphobilinogen deaminase (HemC).

Enrichment of Low Abundance Proteins of Helicobacter pylori Strain 26695 by the Heparin Chromatography

Low-abundance cellular proteins normally invisible on the standard two-dimensional SDS-polyacrylamide gel electrophoresis (2-DE SDS-PAGE) map must be enriched appropriately in order to be visualized and identified in cells or tissues. We applied proteins of H. pylori strain 26695 to a immobilized heparin-affinity resin, which has an affinity for nucleic acid-binding proteins, protein biosynthesis factors, and growth factors. The whole cell extract of H. pylori strain 26695 was fractionated by the heparin-agarose chromatography, and was analyzed by 2-DE. The 2-DE SDS-PAGE displayed spots after silver staining, which were identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Among the ca. 150 spots that were processed, 79 proteins representing 57 genes were identified. Eleven proteins were determined to be nucleic acid-associated. Eighteen proteins were newly identified in this study, including DNA topoisomerase I. These results may provide guidance for enriching low abundance proteins of H. pylori and contribute to the construction of a master protein map of H. pylori.