Abstract
This chapter presents methods for capillary electrophoresis (CE) fingerprinting of proteins in a cell extract and in single cells. A custom-made CE instrument with laser-induced fluorescence (LIF) detection, used for the analyses, is described. Detailed procedures are given for: (1) on column labeling of proteins with a fluorogenic reagent, 5-furoyl quinoline-3- carboxaldehyde, (2) CE separation of labeled proteins, (3) preparation of a protein extract from cultured cells, and (4) manipulations associated with analyses of proteins in single cells. More than 20 relevant publications are cited in this chapter to assist the reader with adopting the presented methods.
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References
Ahmadzadeh, H. and Dovichi, N. J. (2000) Instrumentation, capillary coating and labeling chemistry for capillary electrophoresis with laser-induced fluorescence detection. Ph.D. thesis, Univ. Alberta, Canada, Chs. 1 and 6.
Albin, M., Weinberger, R., Sapp E., and Moring, S. (1991) Fluorescence detection in capillary electrophoresis: evaluation of derivatizing reagents and techniques. Anal. Chem. 63, 417–422.
Wu, S. and Dovichi, N. J. (1989) High-sensitivity fluorescence detector for fluoresceinisothiocyanate derivatives of amino acids separated by capillary electrophoresis. J. Chromatogr. 480, 141–155.
Dovichi, N. J., Martin, J. C., Jett, J. H., Trkula, M., and Keller R. A. (1984) Laser-induced fluorescence of flowing samples as an approach to single-molecule detection in liquids. Anal. Chem. 56, 348–354.
McGuffin, V. L. and Zare, R.N. (1985) Laser fluorescence detection in microcolumn liquid chromatography: application to derivatized carboxylic acids. Appl. Spectrosc. 39, 847–853.
Swerdlow, H., Zhang, J. Z., Chen, D. Y., et al. (1991) Three DNA sequencing methods using capillary gel electrophoresis and laser-induced fluorescence detection. Anal. Chem. 63, 2835–2841.
Lynos, J. W. and Faulkner, L.R. (1982) Optimization of flow cells for fluorescence detection in liquid chromatography. Anal. Chem. 54, 1960–1964.
Zarrin, F. and Dovichi, N. J. (1985) Particle counting by laser light scatter for capillary hydrodynamic chromatography. Anal. Chem. 57, 1826–1829.
Zarrin, F., Risfelt, J. A., and Dovichi, N. J. (1987) Light scatter detection within the sheath-flow cuvette for size determination of multicomponenet submicrometer particle suspensions. Anal. Chem. 59, 850–854.
Zarrin, F., Bornhop, D. J., and Dovichi N. J. (1987) Laser Doppler velocimetry for particle size determination by light scatter within the sheath-flow cuvette. Anal. Chem. 59, 854–860.
Cheng, Y. F. and Dovichi, N. J. (1988) Subattomole amino acid analysis by capillary zone electrophoresis and laser-induced fluorescence. Science 242, 562–564.
Cheng, Y. F., Wu, S., Chen, D. Y., and Dovichi, N. J. (1990) Interaction of capillary zone electrophoresis with a sheath-flow cuvette detector. Anal. Chem. 62, 496–503.
Wu, S. and Dovichi, N. J. (1992) Capillary zone electrophoresis separation and laser-induced fluorescence detection of zeptomole quantities of fluoresceinthiohydantoin derivatives of amino acids. Talanta 39, 173–178.
Chen, D. Y., Swerdlow, H. P., Harke, H. R., Zhang, J. Z., and Dovichi N. J. (1991) Low-cost, high-sensitivity laser-induced fluorescence detection for DNA sequencing by capillary gel electrophoresis. J. Chromatogr. 559, 237–246.
Zhang, J. Z., Chen, D. Y., Wu, S., Harke, H. R., and Dovichi, N. J. (1991) Highsensitivity laser-induced fluorescence detection for capillary electrophoresis. Clin. Chem. 37, 1492–1496.
Camilleri, P. (1993) Capillary Electrophoresis Theory and Practice. CRC, Boca Raton, FL, pp. 25–64.
Haugland, R. P. (1996) Handbook of Fluorescent Probes and Research Chemicals. Molecular Probes, Eugene, OR.
Novotny, M. (1988) Recent advances in microcolumn liquid chromatography. Anal. Chem. 60, 500A–510A.
Liu, J., Hsieh, Y. Z., Wiesler, D., and Novotny, M. (1991) Design of 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde as a reagent for ultrasensitive determination of primary amines by capillary electrophoresis using fluorescence detection. Anal. Chem. 63, 408–412.
Camilleri, P. (1993) Capillary Electrophoresis Theory and Practice. CRC, Boca Raton, FL, pp. 311–370.
Pinto D., Arriaga E. A., Craig D., et al. (1997) Picomolar assay of native proteins by capillary electrophoresis precolumn labeling submicellar separation and laser-induced fluorescence detection. Anal. Chem. 69, 3015–3021.
Zhao J. Y., Waldron K. C., Miller J. Z., Harke H. R., and Dovichi N. J. (1992) Attachment of a single fluorescent label to peptides for determination by capillary zone electrophoresis. J. Chromatogr. 608, 239–242.
Tsuda T., Kobayashi Y., Hori A., Matsumoto T., and Suzuki O. (1988) Post-column detection for capillary zone electrophoresis. J. Chromatogr. 456, 375–381.
Roze D. J. and Jorgenson J. W. (1988) Post-capillary fluorescence detection in capillary zone electrophoresis using o-Phthaldialdehyde. J. Chromatogr. 447, 117–131.
Pentoney S. L., Huang X., Burgi D. S., and Zare R. N. (1988) On-line connector microcolumns: application to the on-column o-Ohthaldialdehyde derivatization of amino acids separated by capillary zone electrophoresis. Anal. Chem. 60, 2625–2629.
Zhang, J. (1994) Ph.D. thesis, Univ. Alberta, Canada.
Krylov, S. N., Starke, D. A., Arriaga, E. A., et al. (2000) Instrumentation for chemical cytometry. Anal. Chem. 72, 872–877.
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Ahmadzadeh, H., Krylov, S. (2004). On-Column Labeling Reaction for Analysis of Protein Contents of a Single Cell Using Capillary Electrophoresis With Laser-Induced Fluorescence Detection. In: Strege, M.A., Lagu, A.L. (eds) Capillary Electrophoresis of Proteins and Peptides. Methods in Molecular Biology™, vol 276. Humana Press. https://doi.org/10.1385/1-59259-798-X:029
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DOI: https://doi.org/10.1385/1-59259-798-X:029
Publisher Name: Humana Press
Print ISBN: 978-1-58829-017-5
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