N Terminal Sequencing Protocol

Edman degradation -- a well-known process for identifying the N-terminal amino acid of a protein -- requires cleavage of the amino acid, followed by identification using chromatography or electrophoresis. Automated processes accelerate N-terminal sequencing.

The Facts

• Proteins produced inside individual cells act as hormones, enzymes and other important regulators. Genetic mutation resulting in an abnormal protein could have serious health consequences.

Process

• The N-terminal amino acid is cleaved by treatment with one of several reagents, then hydrolyzed to form a separate amino acid. Thin layer chromatography, HPLC or electrophoresis determines the size of the amino acid, and compares it to a known size standard. The new N-terminal end of the remaining protein can be similarly cleaved for identification.

Automation

• Applied Biosystems led the development of automating Edman degradation, which binds a purified protein to a membrane and treats it with phenylisothiocyanate. The free amino acid's automatically injected into a HPLC system for identification.

Complications

• Proteins must stay relatively pure and free of contaminants to accurately determine the N-terminal amino acid. Edman degradation limits require large protein molecules fragmented into small peptide chains no longer 50 amino acids.