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Proteolytic Mapping

Peptides and Proteins

  1. Tim Nadler

Published Online: 15 SEP 2006

DOI: 10.1002/9780470027318.a1630

Encyclopedia of Analytical Chemistry

Encyclopedia of Analytical Chemistry

How to Cite

Nadler, T. 2006. Proteolytic Mapping. Encyclopedia of Analytical Chemistry. .

Author Information

  1. PE Biosystems, Framingham, USA

Publication History

  1. Published Online: 15 SEP 2006

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.