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

Soo Han Bae, Andrew G. Harris, Peter G. Hains, Hong Chen, David E. Garfin, Stuart L. Hazell, Young Ki Paik, Bradley J. Walsh, Stuart J. Cordwell

Research output: Contribution to journalArticle

61 Citations (Scopus)

Abstract

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 (p/) 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 p/ of greater than 10.23. A second approach to examine extremely alkaline proteins (p/ > 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 p/ > 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.

Original languageEnglish
Pages (from-to)569-579
Number of pages11
JournalProteomics
Volume3
Issue number5
DOIs
Publication statusPublished - 2003 May 1

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Proteome
Proteins
Liquid chromatography
Liquid Chromatography
Proton-Motive Force
Technology
Electrophoresis
Complex Mixtures
Helicobacter pylori
Mass spectrometry
Complement C2
Gene encoding
Electrophoresis, Gel, Two-Dimensional
Isoelectric Point
Isoelectric Focusing
Pathogens
Human Genome
Tandem Mass Spectrometry
Sodium Dodecyl Sulfate
Ionization

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Bae, S. H., Harris, A. G., Hains, P. G., Chen, H., Garfin, D. E., Hazell, S. L., ... Cordwell, S. J. (2003). Strategies for the enrichment and identification of basic proteins in proteome projects. Proteomics, 3(5), 569-579. https://doi.org/10.1002/pmic.200300392
Bae, Soo Han ; Harris, Andrew G. ; Hains, Peter G. ; Chen, Hong ; Garfin, David E. ; Hazell, Stuart L. ; Paik, Young Ki ; Walsh, Bradley J. ; Cordwell, Stuart J. / Strategies for the enrichment and identification of basic proteins in proteome projects. In: Proteomics. 2003 ; Vol. 3, No. 5. pp. 569-579.
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Bae, SH, Harris, AG, Hains, PG, Chen, H, Garfin, DE, Hazell, SL, Paik, YK, Walsh, BJ & Cordwell, SJ 2003, 'Strategies for the enrichment and identification of basic proteins in proteome projects', Proteomics, vol. 3, no. 5, pp. 569-579. https://doi.org/10.1002/pmic.200300392

Strategies for the enrichment and identification of basic proteins in proteome projects. / Bae, Soo Han; Harris, Andrew G.; Hains, Peter G.; Chen, Hong; Garfin, David E.; Hazell, Stuart L.; Paik, Young Ki; Walsh, Bradley J.; Cordwell, Stuart J.

In: Proteomics, Vol. 3, No. 5, 01.05.2003, p. 569-579.

Research output: Contribution to journalArticle

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Bae SH, Harris AG, Hains PG, Chen H, Garfin DE, Hazell SL et al. Strategies for the enrichment and identification of basic proteins in proteome projects. Proteomics. 2003 May 1;3(5):569-579. https://doi.org/10.1002/pmic.200300392