Resource

Online Inquiry

Workflow of Edman degradation

Pehr Edman published a protein sequencing method, Edman Degradation, in 1950. This method made it possible to determine the extended sequences of peptides or whole proteins and is widely used today. The method removes and identifies amino acid residues at the end of the peptide chain, i.e. residues with free α-amino groups, through a series of chemical reactions. At the same time, the next residue in the sequence is made and subjected to the same round of chemical reactions. Repeating this process reveals the sequence of the polypeptide. This method is a powerful tool for proteomics studies.

Workflow of Edelman degradation

Edelman degradation is mainly divided into several steps such as coupling, cleavage, extraction, conversion and identification.

Coupling of Edman degradation

Phenyl isothiocyanate (also known as PITC) is used to react with protein samples. This reagent reacts with the N-terminal end of the amino group under mild alkaline conditions to form phenylthiocyaninyl (PTC).

PITC structure

PITC structure

Edman degradation cleavage

In the presence of a strong acid, the first peptide bond is cleaved, resulting in a peptide segment that loses the first base and the release of the first residue in the form of anilinothiazolinone (ATZ). Washing off the other reactants and the released residues, the shortened peptide fragment can release the second residue by another round of coupled reaction and cleavage reaction, and so on until the last amino acid residue is released.

Currently, trifluoroacetic acid (TFA) is used for this cleavage reaction. This is because to minimize acid hydrolysis at each site of the polypeptide chain, an anhydrous environment needs to be created as much as possible. A new N-terminal can then be generated, which can then be sequenced. Minimizing this acid cleavage can lead to a more durable and clearer sequence.

Residue conversion of Edman degradation

The ATZ residue was separated from the peptide by extraction with organic solvents (ethyl acetate or chlorobutane), which was then converted to the more stable phenylthiohydantoin (PTH) form. Some modified amino acid residues (eg, glycosylated asparagine groups) may be poorly soluble in organic solvents and therefore appear blank at corresponding points in their sequences. In solid phase sequencing, other methods of extraction of ATZ residues can be attempted when the peptide is covalently attached to a solid support. Note, however, that the remaining peptides cannot be extracted or lost (resulting in a dramatic drop in yield).

Analysis of PTH residues by dman degradation

PTH residues resulting from each cycle of Edman degradation are typically identified using chromatographic methods, initially thin-layer chromatography and later reversed-phase high-performance liquid chromatography. The PTH amino acid residues from each cycle are identified and quantified sequentially by comparison with standards, and the sequences are described in order from the N-terminal to the C-terminal.

Other methods for protein sequencing

Short protein sequences (10 to 15 residues) identified by Edman degradation are translated into DNA sequences and used as probes or primers to isolate molecular clones of the corresponding gene or complementary DNA. The sequence of the cloned DNA is then determined and used to infer the complete amino acid sequence of the protein.

De novo sequencing of proteins is used to infer the amino acid sequence based on the mass difference between a series of regular fragment ions produced by the collision of a peptide with an inert gas. The amino acid sequence as well as post-translational modifications can be inferred from the y and b ions at the peptide bond break. De novo sequencing has an outstanding advantage of de novo sequencing of unknown proteins without relying on any protein database.

Related Resource

Principle, advantages and disadvantaged of Edman sequencing

* For Research Use Only. Not for use in diagnostic procedures.
Our customer service representatives are available 24 hours a day, 7 days a week. Inquiry

Online Inquiry

Please submit a detailed description of your project. We will provide you with a customized project plan to meet your research requests. You can also send emails directly to for inquiries.

* Email
Phone
* Service & Products of Interest
Services Required and Project Description
* Verification Code
Verification Code