Proteolytic Mapping

Abstract

Proteolytic mapping is a technique used to verify the primary structure of a protein. It involves the cleavage of the protein at specific amino acids to generate a unique set of peptides which is then separated and analyzed. The chromatogram generated during the separation is known as the proteolytic map of the protein, which serves as a unique “fingerprint” for that protein. A proteolytic map can be used to verify the identity of a protein and to detect mutations in a protein. This technique is faster than two‐dimensional (2D) nuclear magnetic resonance (NMR), X‐ray crystallography, or complete amino acid sequencing for verifying the primary structure of a protein. However, it does not allow determination of the sequence of a protein de novo as can be accomplished with sequencing, nor does it yield the higher order structural information that may be obtained with 2D NMR or X‐ray crystallography. The process of proteolytic mapping involves sample preparation, enzyme digestion, and map development. Each of these steps should be optimized for each new protein to obtain a good map; alternatively, generic protocols can be used to produce satisfactory results for some proteins. A typical protocol requires about a day to complete owing to the time required to digest a protein with an enzyme. However, more recent automated techniques are capable of completing the process in a few hours because they employ a highly concentrated immobilized enzyme which speeds digestion. Proteolytic maps are used in both quality control (QC) and research environments, with the most recent application in the field of proteomics.