Peptide Mapping by Two-Dimensional Thin-Layer Electrophoresis-Thin-Layer Chromatography

Abstract

The principle behind peptide mapping is straightforward: If two proteins have the same primary structures, then cleavage of each protein with a specific protease or chemical cleavage reagent will yield identical peptide fragments. However, if the proteins have different primary structures, and then the cleavage will generate unrelated peptides. The similarity or dissimilarity of the proteins’ primary structure is reflected in the similarity or dissimilarity of the peptide fragments. Separation of peptides by 2D thin-layer electrophoresis-thin-layer chromatography (2D TLE-TLC) results in very high resolution of the peptides, making subtle comparisons possible. There are four phases to the 2D TLE-TLC peptide mapping process:

• 1. Identification and purification of the proteins to be compared;

• 2. Radiolabeling of the proteins, and thus the peptide fragments, to minimize the quantity of protein required;

• 3. Cleavage of the proteins with specific endopeptidic reagents, either chemical or enzymatic; and

• 4. Separation and visualization of the peptide fragments for comparison.