Overview of Amino Acid N-Terminal Sequencing and Edman Degradation Method
N-terminal amino acid sequencing is a technique used to determine the order of the first few amino acid residues in a protein or polypeptide sequence starting from the N-terminal (amino terminal) end. This method is crucial for protein identification and characterization, particularly in biochemical and molecular biology research.
The Edman degradation method is the most commonly used amino acid N-terminal sequencing technique. This method involves stepwise chemical degradation to remove one amino acid at a time from the sequence, thereby determining the amino acid order of the protein or polypeptide. The process is generally as follows:
1. Label the N-terminal amino acid:
First, a specific chemical reagent (usually Edman reagent, phenyl isothiocyanate) is used to react with the N-terminal amino acid of the polypeptide or protein. This reaction forms a stable labeled compound.
2. Cleave the labeled amino acid:
Next, a special chemical method is used to cleave the labeled amino acid from the polypeptide chain. The cleaved amino acid is a stable derivative that can be analyzed and identified using techniques such as high-performance liquid chromatography (HPLC).
3. Repeat the process:
Then, the process is repeated for the remaining polypeptide or protein, removing a new N-terminal amino acid each time. By gradually repeating this process, the entire sequence of the polypeptide or protein can be determined step by step.
Figure 1. Edman sequencing experimental flowchart
It should be noted that the efficiency of Edman degradation is not 100%, which means that as the sequencing depth increases, the signal gradually weakens. Therefore, this method is typically used to determine only the first 20-30 amino acids of a protein. For long-chain proteins or when the entire sequence needs to be determined, other methods, such as mass spectrometry, are usually combined.
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