Chemical Cleavage of Proteins at Methionyl-X Peptide Bonds

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.