36] Paper chromatography and electrophoresis; special procedure for peptide maps

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The development of procedures for peptide mapping requires a technique that is capable of giving highly valuable information on the primary structure of protein molecules. This chapter discusses the procedure of peptide mapping by using two major techniques, namely––paper chromatography and electrophoresis. An enzymatic digest of a protein to chromatography is subjected, followed by high-voltage electrophoresis in the second dimension. The relative mobility of each peptide in the two dimensions depends on its composition and to a lesser extent its sequence. The procedure is designed to afford a rough identification of differences in amino acid sequence between two closely related molecules, such as residue replacements due to genetic mutation. Several solvent systems are used for peptide mapping and vary in applicability to different proteolytic digests. A variety of amino acid-specific reagents used to stain a peptide map and enhance its usefulness are focused. Many specific staining techniques found in standard texts dealing with chromatography and are helpful in technique of peptide mapping such as Ehrlich stain for indoles are discussed. Interpretation of the map, peptide mapping for preparative purposes, peptide mapping to evaluate digestion by exopeptidases, and technique of diagonal peptide mapping are also discussed in the chapter.