What Is Edman degradation?
N-terminal sequencing by Edman degradation, is a traditional method for sequencing protein, and still has advantages for protein analysis that cannot readily be obtained by other analysis methods. Edman degradation is a cyclic procedure: N-terminal amino acid residues is labeled and cleaved off from the peptides or proteins at a time, and is identified by chromatography. Generally, the N-terminal amino group of the protein is reacted with phenyl isothiocyanate to form a phenylthiocarbamoyl derivative. Under mildly acidic conditions, the derivative is released from the rest of the protein as a cyclic phenylthiohydantoin (PTH) amino acid. The released PTH amino acid is identified by high performance liquid chromatography (HPLC) or HPLC-MS. The remainder of the peptide is intact and available for another round of labeling and release.
What Is the Characteristic of Edman Degradation?
Edman sequencing is not a sensitive method, requiring several picomoles and purified protein, to obtain a define sequence information. One important thing for Edman sequencing is that the α-amino group at the N-terminal end of the molecule should be unmodified before undergoing the cyclic sequencing process. Theoretically, it is possible to obtain sequence of the entire peptide or protein in a single sequencer run. However, multiple factors limit the amount of sequence in practice. With current technology, it is fairly routine to obtain at least 20 to 25 residues of sequence from the N-terminus of the proteins and peptides.
What Is Edman Degradation Used for?
Edman sequencing is usually used to sequence novel proteins which have neither nucleotide nor amino acid sequence in database for matching. Edman sequencing confirms protein N-terminus and cleavage sites. Furthermore, this method is used to verify the N-terminal boundary of recombinant proteins, particularly proteins larger than 40-80 kDa when highly accurate masses cannot be obtained by ESI-MS.
