On-Column Labeling Reaction for Analysis of Protein Contents of a Single Cell Using Capillary Electrophoresis With Laser-Induced Fluorescence Detection

  • Hossein Ahmadzadeh
  • Sergey Krylov
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 276)

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.

Key Words

Capillary electrophoresis labeling laser-induced fluorescence proteins single cell 

References

  1. 1.
    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.Google Scholar
  2. 2.
    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.CrossRefGoogle Scholar
  3. 3.
    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.PubMedCrossRefGoogle Scholar
  4. 4.
    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.PubMedCrossRefGoogle Scholar
  5. 5.
    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.CrossRefGoogle Scholar
  6. 6.
    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.PubMedCrossRefGoogle Scholar
  7. 7.
    Lynos, J. W. and Faulkner, L.R. (1982) Optimization of flow cells for fluorescence detection in liquid chromatography. Anal. Chem. 54, 1960–1964.CrossRefGoogle Scholar
  8. 8.
    Zarrin, F. and Dovichi, N. J. (1985) Particle counting by laser light scatter for capillary hydrodynamic chromatography. Anal. Chem. 57, 1826–1829.PubMedCrossRefGoogle Scholar
  9. 9.
    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.PubMedCrossRefGoogle Scholar
  10. 10.
    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.PubMedCrossRefGoogle Scholar
  11. 11.
    Cheng, Y. F. and Dovichi, N. J. (1988) Subattomole amino acid analysis by capillary zone electrophoresis and laser-induced fluorescence. Science 242, 562–564.PubMedCrossRefGoogle Scholar
  12. 12.
    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.CrossRefGoogle Scholar
  13. 13.
    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.PubMedCrossRefGoogle Scholar
  14. 14.
    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.PubMedCrossRefGoogle Scholar
  15. 15.
    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.PubMedGoogle Scholar
  16. 16.
    Camilleri, P. (1993) Capillary Electrophoresis Theory and Practice. CRC, Boca Raton, FL, pp. 25–64.Google Scholar
  17. 17.
    Haugland, R. P. (1996) Handbook of Fluorescent Probes and Research Chemicals. Molecular Probes, Eugene, OR.Google Scholar
  18. 18.
    Novotny, M. (1988) Recent advances in microcolumn liquid chromatography. Anal. Chem. 60, 500A–510A.PubMedCrossRefGoogle Scholar
  19. 19.
    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.PubMedCrossRefGoogle Scholar
  20. 20.
    Camilleri, P. (1993) Capillary Electrophoresis Theory and Practice. CRC, Boca Raton, FL, pp. 311–370.Google Scholar
  21. 21.
    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.CrossRefGoogle Scholar
  22. 22.
    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.PubMedCrossRefGoogle Scholar
  23. 23.
    Tsuda T., Kobayashi Y., Hori A., Matsumoto T., and Suzuki O. (1988) Post-column detection for capillary zone electrophoresis. J. Chromatogr. 456, 375–381.CrossRefGoogle Scholar
  24. 24.
    Roze D. J. and Jorgenson J. W. (1988) Post-capillary fluorescence detection in capillary zone electrophoresis using o-Phthaldialdehyde. J. Chromatogr. 447, 117–131.Google Scholar
  25. 25.
    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.PubMedCrossRefGoogle Scholar
  26. 26.
    Zhang, J. (1994) Ph.D. thesis, Univ. Alberta, Canada.Google Scholar
  27. 27.
    Krylov, S. N., Starke, D. A., Arriaga, E. A., et al. (2000) Instrumentation for chemical cytometry. Anal. Chem. 72, 872–877.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Hossein Ahmadzadeh
    • 1
  • Sergey Krylov
    • 2
  1. 1.Department of ChemistryUniversity of MinnesotaMinneapolis
  2. 2.Department of ChemistryYork UniversityTorontoCanada

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