Enzymatic Digestion of Membrane-Bound Proteins for Peptide Mapping and Internal Sequence Analysis

  • Joseph Fernandez
  • Sheenah M. Mische
Part of the Springer Protocols Handbooks book series (SPH)


Enzymatic digestion of membrane-bound proteins is a sensitive procedure for obtaining internal sequence data of proteins that either have a blocked amino terminus or require two or more stretches of sequence data for DNA cloning or confirmation of protein identification. Since the final step of protein purification is usually SDS-PAGE, electroblotting to either PVDF or nitrocellulose is the simplest and most common procedure for recovering protein free of contaminants (SDS, acrylamide, and so forth) with a high yield. The first report for enzymatic digestion of a nitrocellulose-bound protein for internal sequence analysis was by Aebersold et al. in 1987, with a more detailed procedure later reported by Tempst et al. in 1990 (1,2). Basically, these procedures first treated the nitrocellulose-bound protein with PVP-40 (polyvinyl pyrrolidone, M r 40,000) to prevent enzyme adsorption to any remaining nonspecific protein binding sites on the membrane, washed extensively to remove excess PVP-40, and the sample was enzymatically digested at 37°C overnight. Attempts with PVDF-bound protein using the above procedures (3,4) give poor results and generally require >25 µg of protein. PVDF is preferred over nitrocellulose because it can be used for a variety of other structural analysis procedures, such as amino-terminal sequence analysis and amino acid analysis. In addition, peptide recovery from PVDF-bound protein is higher, particularly from higher retention PVDF (ProBlott, Westran, Immobilon Psq). Finally, PVDF-bound protein can be stored dry as opposed to nitrocellulose, which must remain wet during storage and work up to prevent losses during digestion.


Amino Acid Analysis Microcentrifuge Tube Peptide Mapping Digestion Buffer Artifact Peak 
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  1. 1.
    Aebersold, R. H., Leavitt, J., Saavedra, R. A., Hood, L. E., and Kent, S. B. (1987) Internal amino acid sequence analysis of proteins separated by one-or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc. Natl. Acad. Sci. USA 84, 6970–6974.PubMedCrossRefGoogle Scholar
  2. 2.
    Tempst, P., Link, A. J., Riviere, L. R., Fleming, M., and Elicone, C., (1990) Internal sequence analysis of proteins separated on polyacrylamide gels at the submicrogram level: improved methods, applications and gene cloning strategies. Electrophoresis 11, 537–553.PubMedCrossRefGoogle Scholar
  3. 3.
    Bauw, G., Van Damme, J., Puype, M., Vandekerckhove, J., Gesser, B., Ratz, G. P., Lauridsen, J. B., and Celis, J. E. (1989) Protein-electroblotting and-microsequencing strategies in generating protein data bases from two-dimensional gels. Proc. Natl. Acad. Sci. USA 86, 7701–7705.PubMedCrossRefGoogle Scholar
  4. 4.
    Aebersold, R. (1993) Internal amino acid sequence analysis of proteins after in situ protease digestion on nitrocellulose, in A Practical Guide to Protein and Peptide Purification for Microsequencing, 2nd ed. (Matsudaira, P., ed.), Academic, New York, pp. 105–154.Google Scholar
  5. 5.
    Fernandez, J., DeMott, M., Atherton, D., and Mische, S. M. (1992) Internal protein sequence analysis: enzymatic digestion for less than 10 µg of protein bound to polyvinylidene difluoride or nitrocellulose membranes. Anal. Biochem. 201, 255–264.PubMedCrossRefGoogle Scholar
  6. 6.
    Fernandez, J., Andrews, L., and Mische, S. M. (1994) An improved procedure for enzymatic digestion of polyvinylidene difluoride-bound proteins for internal sequence analysis. Anal. Biochem. 218, 112–117.PubMedCrossRefGoogle Scholar
  7. 7.
    Fernandez, J., Andrews, L., and Mische, S. M. (1994) A one-step enzymatic digestion procedure for PVDF-bound proteins that does not require PVP-40, in Techniques in Protein Chemistry V (Crabb, J., ed.), Academic Press, San Diego, pp. 215–222.Google Scholar
  8. 8.
    Best, S., Reim, D. F., Mozdzanowski, J., and Speicher, D. W., (1994) High sensitivity sequence analysis using in-situ proteolysis on high retention PVDF membranes and a bi-phasic reaction column sequencer, in Techniques in Protein Chemistry V (Crabb, J., ed.), Academic, New York, pp. 205–213.Google Scholar
  9. 9.
    Atherton, D., Fernandez, J., and Mische, S. M. (1993) Identification of cysteine residues at the 10 pmol level by carboxamidomethylation of protein bound to sequencer membrane supports. Anal. Biochem. 212, 98–105.PubMedCrossRefGoogle Scholar
  10. 10.
    Kirchner, M., Fernandez, J., Agashakey, A., Gharahdaghi, F., and Mische, S. M. (1996) in Techniques in Protein Chemistry VII (Marshak, O., ed.), Academic, New York (in press).Google Scholar
  11. 11.
    Atherton, D., (1989) Successful PTC amino acid analysis at the picomole level, in Techniques in Protein Chemistry (Hugli, T., ed.) Academic, New York, pp. 273–283.Google Scholar
  12. 12.
    Mozdzanowski, J. and Speicher, D. W. (1990) Quantitative electrotransfer of proteins from polyacrylamide gels onto PVDF membranes, in Current Research in Protein Chemistry: Techniques, Structure, and Function (Villafranca, J., ed.), Academic, New York, pp. 87–94.Google Scholar
  13. 13.
    Tiller, G. E., Mueller, T. J., Dockter, M. E., and Struve, W. G. (1984) Hydrogenation of Triton X-100 Eliminates Its Fluorescence and Ultraviolet Light Absorbance while Preserving Its Detergent Properties. Anal. Biochem. 141, 262–266.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1996

Authors and Affiliations

  • Joseph Fernandez
    • 1
  • Sheenah M. Mische
    • 1
  1. 1.Protein/DNA Technology CenterThe Rockefeller UniversityNew York

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