Edman Degradation: Techniques for N-Terminal Amino Acid Sequencing of Proteins and Peptides

The Edman degradation method is a technique used for determining the N-terminal amino acid sequence of small peptides and proteins. This method is based on periodically removing the amino acid at the N-terminus of the peptide chain and detecting its identity. This process can be repeated, thereby consecutively determining the sequence of multiple amino acids.

 

The general steps of Edman degradation are as follows:

 

Selective Labeling of Peptide Bonds

First, the sample reacts with the Edman reagent (phenyl isothiocyanate), which specifically forms a cyclic phenylthiohydantoin derivative with the N-terminal amino acid of the peptide.

 

Cleavage of the Phenylthiohydantoin Derivative

After phenylthiohydantoination, the phenylthiohydantoinated amino acid can be released from the peptide chain under mild acidic conditions (such as trifluoroacetic acid) without breaking the other amino acids on the peptide chain.

 

Amino Acid Detection

The released phenylthiohydantoin amino acid can be separated and detected by chromatography, thereby determining its identity.

 

Repeat the Process

The remaining peptide reacts again with the Edman reagent, releasing the next N-terminal amino acid, and so on.

 

Although this method is useful in determining the sequence of small peptides, it has some limitations. The biggest limitation is the efficiency loss that occurs during the degradation process, which usually makes it difficult to degrade more than 15-20 amino acids consecutively. For larger proteins, it is usually necessary to decompose them into smaller fragments and then perform Edman degradation on these fragments. In recent years, due to the progress of mass spectrometry technology, mass spectrometry has become the main tool for protein sequencing and identification, but in certain situations, Edman degradation is still a useful complementary method.