We defined the term chemical cytometry to refer to the chemical analysis of the content of single cells. In particular, we are interested in the use of capillary electrophoresis and laser-induced fluorescence to monitor the composition and metabolism of single cells. We have several goals in these projects. First, we are funded by the National Cancer Institute of NIH to monitor changes in protein expression in cancer cells. Our hope is that the protein signature will form a fingerprint that can be used as a prognostic indicator for the disease and that can guide therapy. Second, we are funded by the National Institute for Human Genome Research of NIH to develop technology to look at the expression of specific regulatory proteins in single yeast cells at the single copy level. By monitoring expression at the single copy level, we can understand the regulation of proteins expressed at the lowest possible levels. Third, we are funded by the Department of Energy Microbial Cell Project to monitor the protein expression of single tetrads of the radiation-resistant bacterium Deinococcus radiodurans. This organism is target for use in bioremediation of storage facilities at Hanford and other locations. Last, in an unfunded project, we monitored biosynthetic and biodegradation of oligosaccharides in single cancer cells. These oligosaccharides are precursors to a number of important biological structures, including the blood-group cell-surface antigens. In the experiments, the cells were treated with a fluorescently labeled disaccharide. Biosynthesis created a set of larger oligomers, while biodegradation created smaller fragments, ultimately completely digesting the molecule.
        In our single-cell proteome project, we have developed technology to inject a single cancer cell into an electrophoresis capillary, to lyse the cell, to fluorescently label the proteins liberated from the cell, to perform electrophoresis on the protein mixture, and to detect the proteins with our high sensitivity sheath-flow cuvette. We have reported the use of both sub-micellar and SDS-DALT electrophoresis for the separation of proteins from a single cancer cell. SDS-DALT refers to separation of proteins based on their size. Conventionally, SDS-PAGE is used for this separation; however, polyacrylamide is difficult to use in capillaries, and we instead rely on other polymers, including pullulan and polyethyleneglycol.
        In our single-molecule experiment, we have genetically engineered yeast to express proteins that have been tagged with green fluorescent protein (GFP). These GFP-labeled proteins are then separated by capillary electrophoresis and detected by fluorescence. We have observed post-translational modification of these proteins, which is revealed as a mobility shift in the GFP-labeled protein during electrophoresis.
        We are performing similar modifications to Deinococcus, in order to monitor stress genes following exposure to radiation and other stresses, which will serve as markers for radiation exposure. We are modifying our single-cell proteome instrumentation to monitor protein expression from the single tetrads of the organism. This modification requires a significant improvement in sensitivity to deal with the minute amount of protein expressed in the minute bacterium.
        We have published several papers describing our single-cell analyses.

• "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 1. 69-74 (2002).
• "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).
• "Single-cell analysis avoids sample processing bias" S.N. Krylov, E. Arriaga, Z. Zhang, N.W.C. Chan, M.M. Palcic, and N.J. Dovichi, Journal of Chromatography 741, 31-35 (2000).
• "Instrumentation for chemical cytometry" S.N. Krylov, D.A. Starke, E.A. Arriaga, Z. Zhang, N.W.C. Chan, M.M. Palcic, and N.J. Dovichi, Analytical Chemistry 72, 872-877 (2000).
• "Metabolic cytometry: monitoring oligosaccharide biosynthesis in single cells by capillary electrophoresis" S.N. Krylov, E.A. Arriaga, N.W.C. Chan, N.J. Dovichi, M.M. Palcic, Analytical Biochemistry 283, 133-135 (2000).
• "Correlating cell cycle with metabolism in single cells: the combination of image and metabolic cytometry" S.N. Krylov, Z. Zhang, N.W.C. Chan, E. Arriaga, M.M. Palcic, and N.J. Dovichi, Cytometry 37, 15-20 (1999).
• "Single cell studies of enzymatic hydrolysis of a tetramethylrhodamine labeled trisaccharide in yeast" X.C. Le, W. Tan, C. Scaman, A. Szpacenko, E. Arriaga, Y. Zhang, N.J. Dovichi, O. Hindsgaul and M.M. Palcic, Glycobiology 9, 219-225 (1999).