Enzymatic digestion of proteins in solution

Analysis of protein covalent structure is less complex and more accurate when performed on peptides derived from the larger protein. In contrast to acid-promoted total hydrolysis, peptides are typically generated by selective proteolysis, i.e., by specifically cleaving peptide bonds with endoproteases that have varying degrees of specificity. This unit presents a protocol that can be used to generate peptide fragments from intact, undenatured proteins. Fragments can be analyzed directly by mass spectrometry (MS) or, more often, are first separated by reversed-phase HPLC (RP-HPLC) and then analyzed by MS or automated sequencing. Most proteins are resistant to enzymatic proteolysis under nondenaturing conditions or are not soluble in aqueous solution. Digestion procedures performed in the presence of chaotropic agents and SDS are described, and support protocols provide instructions for preparing enzyme stocks and reducing and alkylating peptides prior to sequencing or HPLC analysis.