Unseen Proteome:  Mining Below the Tip of the Iceberg To Find Low Abundance and Membrane Proteins

Susanne K. Pedersen,^† Jenny L. Harry,^† Lucille Sebastian,^† Jasmine Baker,^† Mathew D. Traini,^† John T. McCarthy,^† Abi Manoharan,^† Marc R. Wilkins,^† Andrew A. Gooley,^† Pier Giorgio Righetti,^‡ Nicolle H. Packer,^† Keith L. Williams,^† and Ben R. Herbert*^†

Journal of Proteome Research, 2003, 2 (3), pp 303–311

DOI: 10.1021/pr025588i

Publication Date (Web): March 12, 2003

Copyright © 2003 American Chemical Society

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Abundant and hydrophilic nonmembrane proteins with isoelectric points below pH 8 are the predominant proteins identified in most proteomics projects. In yeast, however, low-abundance proteins make up 80% of the predicted proteome, approximately 50% have pI's above pH 8 and 30% of the yeast ORFs are predicted to encode membrane proteins with at least 1 trans-membrane span. By applying highly solubilizing reagents and isoelectric fractionation to a membrane fraction of yeast we have a purified and identified 780 protein isoforms, representing 323 gene products, including 28% low abundance proteins and 49% membrane or membrane associated proteins. More importantly, considering the frequency and importance of co- and post-translational modifications, the separation of protein isoforms is essential and two-dimensional electrophoresis remains the only technique which offers sufficient resolution to address this at a proteomic level.

Keywords: two-dimensional electrophoresis • membrane proteins • low-abundance proteins • multi-compartment electrolyzer • fractionation