Chemical Cleavage of Proteins at Methionyl-X Peptide Bonds

  • Bryan John Smith
Part of the Springer Protocols Handbooks book series (SPH)


One of the most commonly used methods for proteolysis uses cyanogen bromide to cleave the bond to the carboxy-(C)-terminal side of methionyl residues. The reaction is highly specific, with few side reactions and a typical yield of 90-100%. It is also relatively simple and adaptable to large or small scale. Because methionine is one of the least abundant amino acids, cleavage at that residue tends to generate a relatively small number of peptides of large size—up to 10,000-20,000 Da. For this reason the technique is usually less useful than some other methods (such as cleavage by trypsin) for identification of proteins by mass mapping, which is better done with a larger number of peptides. Cleavage at Met-X can be useful for other purposes, however:

  1. 1.

    Generation of internal sequence data, from the large peptides produced (1).

  2. 2.

    Peptide mapping.

  3. 3.

    Mapping of the binding sites of antibodies (2) or ligands (3).

  4. 4.

    Generation of large, functionally distinct domains (e.g., from hirudin, by Wallace et al. [4] or proteins of interest from fusion proteins (5).

  5. 5.

    Confirmation of estimates of methionine content by amino acid analysis, which has a tendency to be somewhat inaccurate for this residue (6). This is by determination of the number of peptides produced by cleavage at an assumed 100% efficiency.



Ammonium Bicarbonate Cleavage Reaction Cyanogen Bromide Homoserine Lactone Cysteic Acid 
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.


  1. 1.
    Yuan, G, Bin, J. C., McKay, D. J., and Snyder, F. F. (1999) Cloning and characterization of human guanine deaminase. Purification and partial amino acid sequence of the mouse protein. J. Biol. Chem. 274, 8175–8180.PubMedCrossRefGoogle Scholar
  2. 2.
    Malouf, N. N., McMahon, D., Oakeley, A. E., and Anderson, P. A. W. (1992). A cardiac troponin T epitope conserved across phyla. J. Biol. Chem. 267, 9269–9274.PubMedGoogle Scholar
  3. 3.
    Dong, M., Ding, X.-Q., Pinon, D. I., Hadac, E. M., Oda, R. P., Landers, J. P., and Miller, L. J. (1999) Structurally related peptide agonist, partial agonist, and antagonist occupy a similar binding pocket within the cholecystokinin receptor. J. Biol. Chem. 274, 4778–4785.PubMedCrossRefGoogle Scholar
  4. 4.
    Wallace, D. S., Hofsteenge, J., and Store, S. R. (1990). Use of fragments of hirudin to investigate thrombin-hirudin interaction. Eur. J. Biochem. 188, 61–66.PubMedCrossRefGoogle Scholar
  5. 5.
    Callaway, J. E., Lai, J., Haselbeck, B., Baltaian, M., Bonnesen, S. P., Weickman, J., et al. (1993). Antimicrob. Agents Chemother. 17, 1614–1619.Google Scholar
  6. 6.
    Strydom, D. J., Tarr, G. E., Pan, Y.-C, E., and Paxton, R. J. (1992). Collaborative trial analyses of ABRF-91AAA, in Techniques in Protein Chemistry III (Angeletti, R. H., ed.) Academic Press, San Diego, New York, Boston, London, Sydney, Tokyo, Toronto, pp. 261–274.Google Scholar
  7. 7.
    Fontana, A. and Gross, E. (1986) Fragmentation of polypeptides by chemical methods in Practical Protein Chemistry A Handbook (Darbre, A., ed. John Wiley and Sons, Chichester, pp. 67–120.Google Scholar
  8. 8.
    Morrison, J. R., Fidge, N. H., and Greo, B. (1990) studies on the formation, separation, and characterisation of cyanogen bromide fragments of human A1 apolipoprotein. Analyt. Biochem. 186, 145–152.PubMedCrossRefGoogle Scholar
  9. 9.
    Kaiser, R. and Metzka, L. (1999) Enhancement of cyanogen bromide cleavage yields for methionyl-serine and methionyl-threonine peptide bonds. Analyt. Biochem. 266, 1–8.PubMedCrossRefGoogle Scholar
  10. 10.
    Beavis, R. C. and Chait, B. T. (1990) Rapid, sensitive analysis of protein mixtures by mass spectrometry. Proc. Natl. Acad. Sci. USA 87, 6873–6877.PubMedCrossRefGoogle Scholar
  11. 11.
    Caprioli, R. M., Whaley, B., Mock, K. K., and Cottrell, J. S. (1991). Sequence-ordered peptide mapping by time-course analysis of protease digests using laser description mass spectrometry in Techniques in Protein Chemistry II (eAngeletti, R. M., ed.) Academic Press, San Diego, pp. 497–510.Google Scholar
  12. 12.
    Andrews, P. C., Allen, M. M., Vestal, M. L., and Nelson, R. W. (1992) Large scale protein mapping using infrequent cleavage reagents, LD TOF MS, and ES MS, in Techniques in Protein Chemistry II (Angeletti, R. M., ed.) Academic Press, San Diego pp. 515–523.Google Scholar
  13. 13.
    Rosa, J. C., de Oliveira, P. S. L., Garrat, R., Beltramini, L., Roque-Barreira, M.-C., and Greene, L. J. (1999) KM+, a mannose-binding lectin from Artocarpus integrifolia: amino acid sequence, predicted tertiary structure, carbohydrate recognition, and analysis of the beta-prism fold. Protein Science 8, 13–24.PubMedCrossRefGoogle Scholar
  14. 14.
    Wang, M. B., Boulter, D., and Gatehouse, J. A. (1994) Characterisation and sequencing of cDNA clone encoding the phloem protein pp2 of Cucurbita pepo Plant Mol. Biol. 24, 159–170.CrossRefGoogle Scholar
  15. 15.
    Stone, K. L., McNulty, D. E., LoPresti, M. L., Crawford, J. M., DeAngelis, R., and Williams, K. R. (1992). Elution and internal amino acid sequencing of PVDF blotted proteins, in Techniques in Protein Chemistry III (Angeletti, R. M., ed.) Academic Press, San Diego, pp. 23–34.Google Scholar
  16. 16.
    Wadsworth, C. L., Knowth, M. W., Burrus, L. W., Olivi, B. B., and Niece, R. L. (1992) Reusing PVDF electroblotted protein samples after N-terminal sequencing to obtain unique internal amino acid sequence, in Techniques in Protein Chemistry III (Angeletti, R. M., ed.) Academic Press, San Diego, pp. 61–68.Google Scholar
  17. 17.
    Horn, M. and Laursen, R. A. (1973) Solid-phase Edman degradation. Attachment of carboxyl-terminal homoserine peptides to an insoluble resin. FEBS Lett. 36, 285–288.PubMedCrossRefGoogle Scholar
  18. 18.
    Murphy, C. M. and Fenselau, C. (1995) Recognition of the carboxy-terminal peptide in cyanogen bromide digests of proteins. Analyt. Chem. 67, 1644–1645.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Bryan John Smith
    • 1
  1. 1.Celltech, R&DSloughUK

Personalised recommendations