Identifying the major proteome components of Helicobacter pylori strain 26695

Cho, Myung-Je; Jeon, Beong-Sam; Park, Jeongwon; Jung, Tae‐Sung; Song, Jae‐Young; Choi, Yeung Joon; Choi, Sang-Haeng; Park, Seong-Gyu; Park, Jeong-Uck; Choe, Mi-Young; Jung, Seun-Ae; Byun, Eun-Young; Baik, Seung‐Chul; Youn, Hee‐Shang; Ko, Gyung‐Hyuck; Lim, Dongbin; Rhee, Kwang‐Ho

doi:10.1002/1522-2683(200204)23:7/8<1161::aid-elps1161>3.0.co;2-7

Cited by 40 publications

(21 citation statements)

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“…Thirty-seven of the ORFs identified here have been characterized for the first time in H. pylori, with the remainder having been characterized in previous studies [39][40]. These results suggest that basic proteins are best analyzed using a combination of specialized methodologies including narrow range basic IPG 2-DE gels, and prefractionation combined with 2-DE, SDS-PAGE and multidimensional LC-MS/MS.…”

mentioning

confidence: 55%

“…Therefore, we believe that over one-third of the H. pylori proteome will be difficult to resolve using standard 2-DE technology due to the prevalence of predicted proteins with extremely alkaline pI. Very few such basic proteins have been resolved in previous studies of the H. pylori proteome [39][40]. We therefore aimed to specifically examine such proteins using a range of commercially available and novel methodologies.…”

Section: Basic Proteins In the Theoretical H Pylori Proteomementioning

confidence: 99%

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Strategies for the enrichment and identification of basic proteins in proteome projects

et al. 2003

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Two-dimensional gel electrophoresis (2-DE) is currently the method of choice for separating complex mixtures of proteins for visual comparison in proteome analysis. This technology, however, is biased against certain classes of proteins including low abundance and hydrophobic proteins. Proteins with extremely alkaline isoelectric points (pI) are often very poorly represented using 2-DE technology, even when complex mixtures are separated using commercially available pH 6-11 or pH 7-10 immobilized pH gradients. The genome of the human gut pathogen, Helicobacter pylori, is dominated by genes encoding basic proteins, and is therefore a useful model for examining methodology suitable for separating such proteins. H. pylori proteins were separated on pH 6-11 and novel pH 9-12 immobilized pH gradients and 65 protein spots were subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry, leading to the identification of 49 unique proteins. No proteins were characterized with a theoretical pI of greater than 10.23. A second approach to examine extremely alkaline proteins (pI . 9.0) utilized a prefractionation isoelectric focusing. Proteins were separated into two fractions using Gradiflow technology, and the extremely basic fraction subjected to both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and liquid chromatography (LC) -tandem mass spectrometry post-tryptic digest, allowing the identification of 17 and 13 proteins, respectively. Gradiflow separations were highly specific for proteins with pI . 9.0, however, a single LC separation only allowed the identification of peptides from highly abundant proteins. These methods and those encompassing multiple LC 'dimensions' may be a useful complement to 2-DE for 'near-to-total' proteome coverage in the alkaline pH range.

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confidence: 55%

Section: Basic Proteins In the Theoretical H Pylori Proteomementioning

confidence: 99%

Strategies for the enrichment and identification of basic proteins in proteome projects

et al. 2003

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“…Two-dimensional electrophoresis (2-DE) sample preparation was carried out as described previously (10). Cultured cells were lysed using a buffer containing 9. glycerol, 2% DTT, and 0.01% bromophenol blue.…”

Section: Two-dimensional Electrophoresis and Image Analysismentioning

confidence: 99%

“…Of them, 64 proteins like urease beta subunit, 60 kDa chaperonin, and alkyl hydroperoxide reductase had been identified in the previous studies (4,10,18,20,21,23,29,33).…”

Section: Peptide Mass Fingerprinting and Protein Identificationmentioning

confidence: 99%

“…The genome of H. pylori has been determined to contain about 1,590 open reading frames (ORFs) (1,26,32). Up to date, more than 250 have been identified to list as proteome components of H. pylori (4,10,18,20,21,23,29,33), revealing more intensive exploration needed to construct proteomic information of H. pylori. Actually, most spots appeared so crowded in the region of pI 4.5~8.0 that they were inseparable in 2-DE using the broad range IPG strip.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2007

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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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Changing Concepts in the Characterisation of Microbes and the Influence of Mass Spectrometry

Shah¹,

Chilton²,

Rajakaruna³

et al. 2010

Mass Spectrometry for Microbial Proteomics

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Identifying the major proteome components of Helicobacter pylori strain 26695

Cited by 40 publications

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Strategies for the enrichment and identification of basic proteins in proteome projects

Strategies for the enrichment and identification of basic proteins in proteome projects

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

Changing Concepts in the Characterisation of Microbes and the Influence of Mass Spectrometry

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