The proteome is the complement of proteins expressed by an organism, tissue, or cell. Classic proteomic analytical tools rely on the use of two-dimensional gel electrophoresis for separation of complex mixtures into components that are revealed as a smattering of spots across the gel. Proteins of interest are identified by excising the spot from the gel, digesting the protein with a protease, typically trypsin, analyzing the fragments by mass spectrometry, and searching a database for proteins that would generate similar patterns.
       This technology generates useful information for protein identification. However, the technology suffers from several important limitations. Two-dimensional electrophoresis is a tedious, manually intensive procedure. While useful in small-scale projects, manual two-dimensional electrophoresis is unacceptably slow and cumbersome for large proteomic projects. Mass spectrometry, why providing information-rich data that is useful in identifying proteins, suffers from limited sensitivity and modest dynamic range.
       Isotope coded affinity tagging (ICAT) was developed by Ruedi Aebersold, Frank Turechek, and Mike Gelb at the University of Washington as an alternative technology that is particularly well suited for the quantitative analysis of change in protein expression. While ICAT automates the separation stage in protein analysis, it still suffers from limited sensitivity.
       We are developing an alternative set of tools for proteomic analysis. Our technology is based on automated two-dimensional capillary electrophoresis for protein separation and laser-induced fluorescence for detection. In this technology, proteins are separated in the first dimension by capillary SDS-DALT electrophoresis, which resolves proteins based on their size. A fraction from the first capillary is transferred to a second capillary, where the contents are separated based on their sub-micellar electrophoretic mobility. Successive fractions are transferred from the first to the second capillary under computer control, subjecting the entire sample to two-dimensional separation. Fluorescence from the migrating components are detected at the end of the second capillary with our high-sensitivity fluorescence detector. The data can be presented as a gray-scale image that resembles a conventional two-dimensional silver-stained gel. The image below was overexposed to help visualize proteins with lower expression level.

       We have demonstrated exquisite sensitivity in protein analysis by fluorescence detection, and we routinely study zeptomole amounts of proteins. We have demonstrated that this instrumentation can analyze the protein content of a single eukaryotic cell. We have also demonstrated a four-order of magnitude dynamic range for fluorescence detection. Last, we have developed a number of multiple capillary fluorescence detectors for DNA sequence analysis. Those detectors are available for proteomic analysis.
       We envision a fully automated system that will analyze 96 samples in parallel, with single-cell sensitivity, no operator intervention, and a four-order of magnitude dynamic range. There are several publications describing our proteomic technology:

• "Fully automated two-dimensional capillary electrophoresis for high-sensitivity protein analysis" D. Michels, S. Hu, R. Schoenherr, M.J. Eggertson, N.J. Dovichi, Molecular and Cellular Proteomics in press.
• "Capillary SDS-DALT electrophoresis of proteins in a single human cancer cell" S. Hu, L. Zhang, L.M. Cook, N.J. Dovichi, Electrophoresis 22, 3677-3682 (2001).
• "Protein analysis of an individual Caenorhabditis elegans single-cell embryo by capillary electrophoresis" S. Hu, R. Lee, Z. Zhang, S.N. Krylov, and N.J. Dovichi Journal of Chromatography B 752, 307-310 (2001).
• "One-dimensional protein analysis of an HT29 human colon adenocarcinoma cell" Z. Zhang, S. Krylov, E.A. Arriaga, R. Polakowski, and N.J. Dovichi Analytical Chemistry 72, 318-322 (2000).
• "Picomolar analysis of proteins using electrophoretically mediated microanalysis and capillary electrophoresis with laser induced fluorescence detection" I.H. Lee, D. Pinto, E.A. Arriaga, Z. Zhang, and N.J. Dovichi, Analytical Chemistry 70, 4546-4548 (1998).