Principle and Reaction Characteristics of Edman Chemical Degradation
The Edman degradation method was first developed by Pehr Edman and is a commonly used method for analyzing the N-terminal amino acid sequence of peptide chains or proteins. The reaction principle and characteristics are described as follows.
The principle of the Edman degradation method is that phenyl isothiocyanate (PITC) couples with the α-amino group at the protein's N-terminus under weak alkaline (TMA) conditions to form phenylthiocarbamoyl peptide (PTC-AA). Then, under anhydrous strong acid (TFA) conditions, the first residue at the N-terminus is cleaved from the complete polypeptide protein chain in the form of 2-phenylthiazolinone (ATZ-AA). Subsequently, under mild acidic conditions, ATZ-AA is converted into a more stable phenylthiourea derivative, PTH-AA. The resulting PTH-AA is sent to high-performance liquid chromatography for analysis to identify the type of amino acid. Repeating the above amino acid identification allows for the sequential analysis of the amino acid sequence from the N-terminus to the C-terminus. For longer protein sequences (polypeptides), the sample can be cut into smaller peptide fragments, analyzed individually, and then pieced together to obtain the complete protein sequence information.
The Edman degradation method uses the labeling reagent phenyl isothiocyanate (PITC), and its advantage is that it does not destroy the remaining polypeptide chain, allowing for repetitive sequencing. Edman degradation N-terminal sequencing can directly analyze the N-terminal sequence of protein polypeptide samples without relying on protein databases (including those with N-terminal mutations or modifications). Therefore, it can be used for N-terminal sequence analysis of novel proteins and polypeptides without database information. Additionally, this method requires a small sample amount (only 50-100 pmol), has high sensitivity, and provides more accurate results. Therefore, using the classical Edman degradation method for N-terminal sequence analysis is relatively common in academic research. However, when the N-terminal is chemically modified or encounters non-α-amino acids, Edman degradation cannot be used for sequencing. The Edman degradation method has the drawbacks of low throughput (generally determining about 50 amino acid sequences at the protein's N-terminus) and the inability to analyze multiple proteins simultaneously.
PTM Biolabs uses the Shimadzu Edman sequencing system to provideN-terminal protein sequencing based on Edman degradationservices for researchers and scientific customers. With our sequencing system, the sequence information of 30 amino acids at the N-terminus can be determined. Using a specific protein loading system, 60-70 amino acids at the N-terminus can be determined. PTM Biolabs has also established a platform for N-terminal sequencing using advanced LC-MS/MS technology, which can identify blocked and modified protein termini, forming a complement withN-terminal protein sequencing based on Edman degradationto ensure the smooth progress of N-terminal sequencing services.
Related services:
Protein N/C-terminal sequencing
Biopharmaceutical N/C-terminal sequencing
Full protein sequence determination
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