Principle of Edman Degradation in Protein Sequencing

Edman Degradation is a classical chemical method used to sequentially identify the amino acids of a polypeptide chain. Since its invention, Edman Degradation has been extensively used in protein chemistry and molecular biology research.

 

Basic Principles of Edman Degradation

Edman Degradation, proposed by Swedish chemist Pehr Edman in 1950, involves labeling and sequentially removing the N-terminal amino acids of a polypeptide to determine its sequence. The process includes the following steps:

 

1. Labeling the N-Terminal Amino Acid

Initially, phenylisothiocyanate (PITC) reacts with the N-terminal amino acid of the polypeptide to form a soluble cyclic derivative known as a phenylthiocarbamyl (PTC) derivative.

 

2. Cleaving the N-Terminal Amino Acid

Subsequently, under acidic conditions, the cyclic reaction converts the N-terminal PTC derivative to a phenylthiohydantoin (PTH) derivative, shortening the polypeptide chain by one amino acid.

 

3. Identifying the PTH Derivative

The resulting PTH derivative is then identified using High-Performance Liquid Chromatography (HPLC) or Gas Chromatography (GC), determining the amino acid type.

 

4. Repetitive Cycles

The remaining polypeptide chain undergoes these steps repeatedly, progressively identifying each N-terminal amino acid until the entire sequence is determined.

 

Advantages and Limitations of Edman Degradation

1. Advantages

(1) High Accuracy: Edman Degradation provides high accuracy in determining polypeptide sequences through the step-by-step cleavage and identification of each amino acid.

(2) Low Sample Requirement: Compared to other protein sequencing methods, Edman Degradation requires relatively smaller sample amounts, making it suitable for analyzing trace samples.

 

2. Limitations

(1) Sample Length Restrictions: This method is less effective for long sequences (typically over 50 amino acids) due to decreasing reaction efficiency with increased cycles.

(2) N-terminal Blockage: If the polypeptide's N-terminal is chemically modified or blocked, Edman Degradation cannot effectively identify the amino acid at that position.

(3) Complex Sample Processing: Processing complex samples may require pre-separation and purification of proteins, adding to the experimental steps and complexity.

 

MtoZ Biolabs provides integrate protein sequencing service by Edman degradation.