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Enzymatic cleavage and HPLC peptide mapping of proteins

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Abstract

Detailed procedures are described for successfully digesting reasonably small quantities (i.e., usually >10 pmol) of proteins with a variety of proteases and for then isolating the resulting peptides by reversephase HPLC. Since sodium dodecyl sulfate-polyacrylamide, gel electrophoresis (SDS-PAGE) appears to be the current method of choice for final purification of proteins for structural analysis, special attention is given to carrying out in-gel proteolytic digests on SDS-PAGE-separated proteins that have usually been stained with Coomassie Blue. A compilation of data from nearly 200 “unknown” samples is used to help provide realistic expectations with respect to the results that are likely to be obtained from carrying out in-gel proteolytic digests on large numbers of proteins.

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References

  1. Kawasaki, H., Emori, Y., and Suzuki, K. (1990) Production and separation of peptides from proteins stained with Coomassie brilliant blue R-250 after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Anal. Biochem. 191, 332–336.

    Article  PubMed  CAS  Google Scholar 

  2. Rosenfeld, J., Capdevielle, J., Guillemot, J., and Ferrara, P. (1992) In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis.Anal. Biochem. 203, 173–179.

    Article  PubMed  CAS  Google Scholar 

  3. Hellman, U., Wernstedt, C., Gonez, J., and Heldin, C. (1995) Improvement of an “in-gel” digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing.Anal. Biochem. 224, 451–455.

    Article  PubMed  CAS  Google Scholar 

  4. Williams, K. R. and Stone, K. L. (1995) In gel digestion of SDS PAGE-separated proteins: observations from internal sequencing of 25 proteins, inTechniques in Protein Chemistry VI (Angeletti, R., ed.), Academic, New York, pp. 143–152.

    Chapter  Google Scholar 

  5. Stone, K. L. and Williams, K. R. (1996) Enzymatic digestion of proteins in solution and in SDS polyacrylamide gels, inThe Protein Protocols Handbook (Walker, J. M., ed.), Humana, Totowa, NJ, pp. 415–425.

    Google Scholar 

  6. 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.

    Article  PubMed  CAS  Google Scholar 

  7. Fernandez, J., DeMott, M., Atherton, D., and Mische, S. M. (1992) Internal protein sequence analysis: enzymatic digestion for less than 10 micrograms of protein bound to polyvinylidene difluoride or nitrocellulose membranes.Anal. Biochem. 201, 255–264.

    Article  PubMed  CAS  Google Scholar 

  8. Stone, K. L., McNulty, D. E., LoPresti, M. B., Crawford, J. M., DeAngelis, R., and Williams, K. R. (1992) Elution and internal amino acid sequencing of PVDF-blotted proteins, inTechniques in Protein Chemistry III (Angeletti, R., ed.), Academic, New York, pp. 23–34.

    Google Scholar 

  9. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227, 680.

    Article  PubMed  CAS  Google Scholar 

  10. Stone, K. L. and Williams, K. R. (1993) Enzymatic digestion of proteins and HPLC peptide isolation, inA Practical Guide to Protein and Peptide Purification for Microsequencing (Matsudaira, P. T., ed.), Academic, New York, pp. 45–69.

    Google Scholar 

  11. Stone, K. L., Elliott, J. I., Peterson, G., McMurray, W., and Williams, K. R. (1990) Reversed-phase high-performance liquid chromatography for fractionation of enzymatic digests and chemical cleavage products of proteins, inMethods in Enzymology (McCloskey, J., ed.), Academic, New York, pp. 389–412.

    Google Scholar 

  12. Sun, Y., Zhou, Z., and Smith, D. (1989) Location of disulfide bonds in proteins by partial acid hydrolysis and mass spectrometry, inTechniques in Protein Chemistry (Hugli, T., ed.), Academic, New York, pp. 176–185.

    Google Scholar 

  13. Stone, K. L., LoPresti, M. B., and Williams, K. R. (1990) Enzymatic digestion of proteins and HPLC peptide isolation in the sub-nanomole range, inLaboratory Methodology in Biochemistry: Amino Acid Analysis and Protein Sequencing (Fini, C., Floridi, A., Finelli, V. N., and Wittman-Liebold, B., eds.), CRC, Boca Raton, FL, pp. 181–205.

    Google Scholar 

  14. Glocker, M. O., Arbogast, B., Schreurs, J., and Deinzer, M. L. (1993) Assignment of the inter- and intramolecular disulfide linkages in recombinant human macrophage colony stimulating factor using fast atom bombardment mass spectrometry.Biochemistry 32, 482–488.

    Article  PubMed  CAS  Google Scholar 

  15. Lombard-Platet, P. and Jalinot, P. (1993) Funnel-well SDS-PAGE: a rapid technique for obtaining sufficient quantities of low-abundance proteins for internal sequence analysis.BioTechniques 15, 669–672.

    Google Scholar 

  16. Chial, H. J. and Splittergerber, A. G. (1993) A comparison of the binding of Coomassie brilliant blue to proteins at low and neutral pH.Anal. Biochem. 213, 362–369.

    Article  PubMed  CAS  Google Scholar 

  17. Williams, K. R., Samandar, S. M., Stone, K. L., Saylor, M., and Rush, J. (1996) Matrix assisted-laser desorption ionization mass spectrometry as a complement to internal protein sequencing, inThe Protein Protocols Handbook (Walker, J. M., ed.), Humana Press, Totowa, New Jersey, pp. 541–555.

    Google Scholar 

  18. Williams, K. R., LoPresti, M., and Stone, K. L. (1996) Internal protein sequencing of SDS-PAGE-separated proteins: optimization of an in gel digest protocol, inTechniques in Protein Chemistry VIII (Marshak, D., ed.), Academic, New York, in press.

    Google Scholar 

  19. Williams, K., LoPresti, M., and Stone, K. (1997) Internal protein sequencing of SDS-PAGE-separated proteins: optimization of an in gel digest protocol, inTechniques in Protein Chemistry VIII (Marshak, D.R. ed.), Academic, San Diego, CA, pp. 79–90.

    Chapter  Google Scholar 

  20. Stone, K. L., LoPresti, M. B., Crawford, J. M., DeAngelis, R., and Williams, K. R. (1991) Reversephase HPLC separation of sub-nanomole amounts of peptides obtained from enzymatic digests, inHigh Performance Liquid Chromatography of Peptides and Proteins: Separation, Analysis and Conformation (Mant, C. T. and Hodges, R. S., eds.), CRC Press, Boca Raton, FL, pp. 669–677.

    Google Scholar 

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Authors and Affiliations

  1. W. M. Keck Foundation Biotechnology Resource Laboratory and Howard Hughes Medical Institute Biopolymer Facility, Yale University, 06536, New Haven, CT

    Kenneth R. Williams & Kathryn L. Stone

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  1. Kenneth R. Williams
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  2. Kathryn L. Stone
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Correspondence to Kenneth R. Williams.

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Williams, K.R., Stone, K.L. Enzymatic cleavage and HPLC peptide mapping of proteins. Mol Biotechnol 8, 155–167 (1997). https://doi.org/10.1007/BF02752260

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