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Reverse-Phase HPLC Isolation and Microsequence Analysis

  • John E. Shively
Protocol
Part of the Biological Methods book series (BM)

Abstract

It is well recognized that reverse-phase HPLC is one of the most powerful methods for peptide and protein isolation. The combination of high resolution and peak sensitivity with detection of the peptide bond at 200–220 nm has made this system an attractive complement to microsequence analysis. Although it is possible to perform reverse-phase HPLC using a variety of solvent systems, the trifluoroacetic acid/acetonitrile (TFA/MeCN) system in particular has become the most widely used because: (1) it has low absorbance at the 200–220 nm range; (2) the recovery of peptides is in the 90–100% range; (3) depending on the column and the protein, the recovery of proteins is also high; (4) peak shape and resolution are superior to the non-ion-pairing systems; and (5) the solvent is volatile and does not interfere with subsequent microsequence analysis.

Keywords

Amino Acid Analysis Initial Yield Peptide Bond Cleavage Affinity Label Peak Recovery 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Böhlen, P., Stein, S., Stone, J., and Udenfriend, S. (1975) Anal. Biochem. 67, 438–445.PubMedCrossRefGoogle Scholar
  2. 2.
    Titani, K., Sasagawa, T., Resing, K., and Walsh, K. A. (1982) Anal. Biochem. 123, 408–412.PubMedCrossRefGoogle Scholar
  3. 3.
    Tempst, P., Hunkapiller, M. W., and Hood, L. E. (1984) Anal. Biochem. 137, 188–195.PubMedCrossRefGoogle Scholar
  4. 4.
    Lewis, R. V., Fallon, A., Stein, S., Gibson, K. D., and Udenfriend, S. (1980) Anal. Biochem. 104, 153–159.PubMedCrossRefGoogle Scholar
  5. 5.
    Haniu, M., Iyanagi, T., Legesse, K., and Shively, J. E. (1984) J. Biool Chem. 259, 13703–13711.Google Scholar
  6. 6.
    Akhrem, A. A., Vasilevsky, V. I., Adamovich, T. B., Labko, A. G., Shkumatov, V. M., and Chashchin, V. L. (1980), in Biochemistry, Biophysics and Regulation of Cytochrome P-450 (Gustafsson, J.-A., Carlstedt-Duke, J. C., Mode, A., and Rafter, J., eds.), pp. 57–64, North-Holland Biomedical Press, Elsevier.Google Scholar
  7. 7.
    Yuan, P.-M., Ryan, D. E., Levin, W., and Shively, J. E. (1983) Proc. Natl. Acad. Sci. USA 80, 1169–1173.PubMedCrossRefGoogle Scholar
  8. 8.
    Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., and Muramatsu, M. (1982) Proc. Natl. Acad. Sci. USA 79, 2793–2797.PubMedCrossRefGoogle Scholar
  9. 9.
    Thomas, P. E., Reik, L. M., Ryan, D. E., and Levin, W. (1983) J. Biol. Chem. 258, 4590–4598.PubMedGoogle Scholar
  10. 10.
    Botelho, L. H., Ryan, D. E., and Levin, W. (1979) J. Biol. Chem. 254, 5635–5640.PubMedGoogle Scholar
  11. 11.
    Botelho, L. H., Ryan, D. E., Yuan, P.-M., Kutny, R., Shively, J. E., and Levin, W. (1982) Biochem. 21, 1152–1155.CrossRefGoogle Scholar
  12. 12.
    Haniu, M., Yuan, P.-M., Ryan, D. E., Levin, W., and Shively, J. E. Biochem. 23, 2478–2482.Google Scholar
  13. 13.
    Haniu, M., Ryan, D. E., Levin, W., and Shively, J. E. (1984) Proc. Natl. Acad. Sci. USA 81, 4298–4301.PubMedCrossRefGoogle Scholar
  14. 14.
    Kawajiri, K., Gotoh, O., Sogawa, K., Tagashira, Y., Muramatsu, M., and Fujii-Kuriyama, Y. (1984) Proc. Natl. Acad. Sci. USA 81, 1649–1653.PubMedCrossRefGoogle Scholar
  15. 15.
    Mort, A. J. and Lamport, D. T. A. (1977) Anal. Biochem. 82, 289–309.PubMedCrossRefGoogle Scholar
  16. 16.
    Edge, A. S. B., Faltynek, C. R, Hof, L., Reichert, L. E., Jr., and Weber, P. (1981) Anal. Biochem. 118, 131–137.PubMedCrossRefGoogle Scholar
  17. 17.
    Glassman, J. N. S., Todd, C. W., and Shively, J. E. (1978) Biochem. Biophys. Res. Comm. 85, 209–216.PubMedCrossRefGoogle Scholar
  18. 18.
    Ozols, J. and Gerard, C. (1977) J. Biol. Chem. 252, 5986–5989.PubMedGoogle Scholar
  19. 19.
    Inglis, A., McKern, N., Roxburgh, C., and Strike, P. (1980) in Methods in Peptide and Protein Sequence Analysis (C. Birr, ed.), pp. 329–343, North-Holland Biomedical Press, Elsevier.Google Scholar
  20. 20.
    Clark, B. R., Zaia, J.A., Balce-Directo, L., and Ting, Y.-P. (1984) J. Viol. 49, 279–282.Google Scholar
  21. 21.
    Pande, H., Baak, S. W., Riggs, A. D., Clark, B.R., Shively, J. E., and Zaia, J. A. (1984) Proc. Natl. Acad. Sci. USA 81, 4965–4969.PubMedCrossRefGoogle Scholar
  22. 22.
    Farley, R. A., Tran, C. M., Carilli, C. T., Hawke, D., and Shively, J. E. (1984) J. Biol. Chem. 259, 9532–9535.PubMedGoogle Scholar
  23. 23.
    Kimmel, J. R., Pollock, H. G., Chance, R. E., Johnson, M. G., Reeve, J. R., Jr., Taylor, I. L., Miller, C., and Shively, J. E. (1984) Endocrinology 114, 1725–1731.PubMedCrossRefGoogle Scholar
  24. 24.
    Reeve, J. R., Jr., Walsh, J. H., Tompkins, R. K., Hawke, D., and Shively, J. E. (1984) Biochem. Biophys. Res. Comm. 123, 404–409.PubMedCrossRefGoogle Scholar
  25. 25.
    Yoo, O. J., Powell, C. T., and Agarwal, K. L. (1982) Proc. Natl. Acad. Sci. USA 79, 1049–1053.PubMedCrossRefGoogle Scholar
  26. 26.
    Podell, D. N. and Abraham, G. N. (1978) Biochem. Biophys. Res. Comm. 81, 176–185.PubMedCrossRefGoogle Scholar
  27. 27.
    Pan, Y.-C. E., Wideman, J., Blacher, R., Chang, M., and Stein, S. (1984) J. Chromat. 297, 13–19.CrossRefGoogle Scholar

Copyright information

© The Humana Press Inc. 1986

Authors and Affiliations

  • John E. Shively
    • 1
  1. 1.Beckman Research Institute of the City of HopeDuarte

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