Abstract
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.
Generation of internal sequence data, from the large peptides produced (1).
-
2.
Peptide mapping.
-
3.
Mapping of the binding sites of antibodies (2) or ligands (3).
-
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.
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.
Keywords
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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
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.
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.
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.
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.
Callaway, J. E., Lai, J., Haselbeck, B., Baltaian, M., Bonnesen, S. P., Weickman, J., et al. (1993). Antimicrob. Agents Chemother. 17, 1614–1619.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Murphy, C. M. and Fenselau, C. (1995) Recognition of the carboxy-terminal peptide in cyanogen bromide digests of proteins. Analyt. Chem. 67, 1644–1645.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2002 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Smith, B.J. (2002). Chemical Cleavage of Proteins at Methionyl-X Peptide Bonds. In: Walker, J.M. (eds) The Protein Protocols Handbook. Springer Protocols Handbooks. Humana Press. https://doi.org/10.1385/1-59259-169-8:485
Download citation
DOI: https://doi.org/10.1385/1-59259-169-8:485
Publisher Name: Humana Press
Print ISBN: 978-0-89603-940-7
Online ISBN: 978-1-59259-169-5
eBook Packages: Springer Book Archive