Enzymatic Digestion of Proteins in Solution and in SDS Polyacrylamide Gels

Abstract

Although most prokaryotic proteins have free NH₂-termini and therefore can be directly sequenced, most eukaryotic proteins have blocked NH₂-termini which precludes Edman degradation (1,2). In these instances, one of the most direct approaches to obtaining partial amino acid sequences is via enzymatic or chemical cleavage followed by peptide fractionation and sequencing. Although several different approaches may be taken to cleave proteins, one of the most common is to digest the protein enzymatically with a relatively specific protease such as trypsin or lysyl endopeptidase. Since final purification is often dependent on SDS-PAGE, cleavage procedures that can either be carried out in the polyacrylamide gel matrix (3,4) or that may be used on samples that have been blotted from SDS polyacrylamide gels onto PVDF (5) or nitrocellulose (5,6) membranes are extremely useful. In both instances, the proteins are usually stained with Coomassie blue or Ponceau S prior to excision, and proteolytic digestion and the resulting peptides are separated by reverse-phase HPLC. Relatively straightforward solution and in-gel digestion procedures that have been used extensively in the Keck Foundation Biotechnology Resource Laboratory at Yale University will be described in this chapter, whereas a procedure suitable for in situ digestion of SDS-PAGE blotted proteins is described in Chapter 70.