Thin-layer liquid culture technique is feasible, and can serve as a versatile liquid culture technique for investigating bacterial properties of H. pylori.
Among 1590 ORFs in the Helicobacter pylori genome, >250 have been identified as authentic genes by proteomic analysis. Low-abundance proteins need to be enriched to a minimal amount for MALDI-TOF analysis and salt precipitation has generally been used for protein enrichment. Here, a whole-cell extract of H. pylori strain 26695 was subjected to protein fractionation with stepwise concentrations of ammonium sulfate and the proteins were displayed by 2-DE. The protein spots were quantified using PDQUEST software and identified by peptide fingerprinting. The 2-DE profiles and intensities of individual protein spots differed among the protein fractions. Out of the 98 identified proteins, 61 were found in the stepwise ammonium sulfate fractions but not in the whole-cell extract. Out of these, 37 proteins, including KdsA, were found exclusively in a single fraction. In contrast, GroEL, UreA, UreB, TrxA, NapA, and FldA were ubiquitously present in all fractions. Iron-containing proteins such as NapA, SodB, CeuE, and Pfr were found predominantly in the 100% saturated ammonium sulfate precipitate. Additionally, 29 proteins were newly identified in this study. These data will facilitate the preparation of significant H. pylori proteins, as well as provide information about low-abundance proteins.
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.
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