Cleaving the polypeptide into smaller fragments

Many polypeptides have a primary structure composed of >100 amino acids. Such molecules cannot be sequenced directly from end to end.
However, these large molecules can be cleaved at specific sites and the resulting fragments sequenced. By using more than one cleaving agent (enzymes and/or chemicals) on separate samples of the purified polypeptide, overlapping fragments can be generated that permit the proper ordering of the sequenced fragments, thereby providing a complete amino acid sequence of the large polypeptide (Fig. 1). Enzymes that hydrolyze peptide bonds are termed peptidases (proteases). [Note: Exopeptidases cut at the ends of proteins and are divided into aminopeptidases and carboxypeptidases. Carboxypeptidases are used in determining the Cterminal amino acid. Endopeptidases cleave within a protein.]

Figure 1. Overlapping of peptides produced by the cleavage action of trypsin and cyanogen bromide.