There are three main reasons why a protein chemist might wish to cleave a protein of interest into peptide fragments. The first reason is to generate, byextensiveproteolysis, a large number of relatively small (5–20 residues) peptides either for peptide mapping (seevol. 1, Chapter 5 ) or for purification and subsequent manual sequence determination by the dansyl-Edman method (seevol. 1, Chapter 24). The second reason is to generate relatively large peptides (50–150 residues) bylimitedproteolysis for automated sequence analysis, such as with the gas-phase sequencer. The third reason is to prepare, again by limited proteolysis, specific fragments for studies relating structure to function, In each case, the specificity of the enzyme used to generate the peptides is a prime consideration, since the aim is to provide high yields of discrete fragments. It can be appreciated that significantly <100% cleavage at some or all of the cleavage sites on the protein being digested will generate a far more complex mixture of a larger number of polypeptides, each in relatively low yield. It is for this reason that enzymes of high specificity, such as trypsin, which cleaves at the C-terminal side of arginine and lysine residues, are mainly used for peptide production.
