Edman Degradation Design Method
Edman degradation is a chemical method used to identify the primary structure of proteins. It is a technique for determining the sequence of proteins, polypeptides, or amino acid residues.
By optimizing the design method of Edman degradation in advance, the success rate of protein sequencing experiments can be improved to a certain extent. It is a very important step in the process of protein sequencing. The specific design methods of Edman degradation are mainly divided into the following parts.
Protein cleavage process: Edman degradation cannot sequence peptides longer than 60 amino acids, so for sequencing long peptides, it is necessary to cleave the long peptide into shorter peptides within the sequencing range of Edman degradation. This cleavage process can be carried out using endopeptidases (such as trypsin or pepsin) or chemical reagents (such as cyanogen bromide). Then, the shorter peptides are sequenced in order, and the sequence information of the entire protein is assembled through overlapping fragments.
Edman reaction process: The protein to be sequenced can be adsorbed onto a solid surface. A common solid substrate is glass fiber coated with polybutadiene (a cationic polymer). Add Edman reagent phenyl isothiocyanate (PITC) together with a weak alkaline buffer solution (pH=8.0-9.0, such as 12% trimethylamine) to the adsorbed protein to react with the α-free amino group at the N-terminus of the amino acid, forming the phenylthiohydantoin (PTC) derivative of the terminal residue, namely PTC-peptide.
Acid cleavage: Anhydrous acid (such as trifluoroacetic acid) is added to selectively separate the N-terminal amino acid. At this time, the PTC derivative undergoes isomerization to generate a replacement, phenylthiohydantoin, which can be detected by eluting the replacement and conducting chromatographic identification to detect the obtained amino acid residues.
Protein identification: Repeat the above chemical reaction cycle, and the amino acids released from the N-terminus of the protein or peptide in each cycle can be identified by chromatographic methods or high-performance liquid chromatography (HPLC).
Biotech company Betopac uses the Shimadzu Edman sequencing system to provide N-terminal protein sequencing based on Edman degradation services for researchers and scientific clients. Using our sequencing system, the sequence information of the N-terminal 30 amino acids can be determined. With a specific protein loading system, the N-terminal 60-70 amino acids can be sequenced. Betopac Biotech has also established a platform for N-terminal sequencing using advanced LC-MS/MS technology, which can determine closed and modified protein termini, complementing the N-terminal protein sequencing based on Edman degradation to ensure the smooth progress of the N-terminal sequencing service.
Related services:
Protein N/C-terminal sequencing
Biopharmaceutical N/C-terminal sequencing
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