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

Bae, Soo-Han and Harris, Andrew G. and Hains, Peter G. and Chen, Hong and Garfin, David E. and Hazell, Stuart L. and Paik, Young-Ki and Walsh, Bradley J. and Cordwell, Stuart J. (2003) Strategies for the enrichment and identification of basic proteins in proteome projects. Proteomics, 3 (5). pp. 569-579. ISSN 1615-9853

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 (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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Item Type: Article (Commonwealth Reporting Category C)
Refereed: Yes
Item Status: Live Archive
Additional Information: Author version not held.
Depositing User: Ms Debbie White
Faculty / Department / School: Historic - Faculty of Sciences - Department of Biological and Physical Sciences
Date Deposited: 09 May 2010 12:45
Last Modified: 02 Jul 2013 23:51
Uncontrolled Keywords: helicobacter pylori; iquid chromatography; mass spectrometry; refractionation; two-dimensional gel electrophoresis
Fields of Research (FOR2008): 06 Biological Sciences > 0605 Microbiology > 060501 Bacteriology
06 Biological Sciences > 0601 Biochemistry and Cell Biology > 060109 Proteomics and Intermolecular Interactions (excl. Medical Proteomics)
Socio-Economic Objective (SEO2008): E Expanding Knowledge > 97 Expanding Knowledge > 970106 Expanding Knowledge in the Biological Sciences
Identification Number or DOI: doi: 10.1002/pmic.200300392
URI: http://eprints.usq.edu.au/id/eprint/7842

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