Main Principle of Edman Sequencing
Edman degradation is the process of determining the sequence of amino acid residues from the N-terminal of a protein polypeptide chain. It achieves the identification of N-terminal amino acids of proteins/polypeptides through a series of chemical reactions including coupling-cyclization cleavage-conversion-PTH-amino acid identification. Understanding the main principles of Edman degradation sequencing has guiding significance for the analysis of the orderly amino acid composition of proteins.
Coupling: In an alkaline environment, the Edman reagent phenyl isothiocyanate (PITC) reacts with the α-amino group of the N-terminal of the protein or polypeptide chain to form the phenylthiocarbamoyl (PTC) derivative of the terminal residue, known as PTC-peptide.
Cyclization cleavage: The coupled product (PTC-peptide) is treated with anhydrous trifluoroacetic acid (TFA), selectively cleaving the first peptide bond at the N-terminal of the polypeptide or protein, releasing the thiazolinone derivative of the amino acid residue (ATZ). Treating PITC-modified peptides with TFA leads to intramolecular cyclization, where the thiourea of the PITC adduct reacts with the carbonyl component of the first peptide bond. This cyclization releases the PITC conjugated with the first amino acid, forming a new N-terminal amine for cyclic reaction.
Conversion: ATZ derivatives are lipophilic and can be extracted using organic solvents (ethyl acetate or chlorobutane), separating the ATZ residue from the peptide. Under acidic conditions, only the amino-terminal residue of the polypeptide chain is liberated as a phenylthiohydantoin (PTH) derivative, which is a phenylthiohydantoin amino acid (PTH-amino acid).
PTH-amino acid identification: Each cycle reaction releases and derivatizes a PTH-amino acid from the N-terminal of the protein or peptide, which is then eluted by high-performance liquid chromatography (HPLC) and identified based on retention time. By comparing with standards, the sequence of each peptide fragment is determined from the N-terminal to the C-terminal after the first amino-terminal residue is liberated, thereby constructing the amino acid sequence of the N-terminal of the protein.
Bio-Techne employs Shimadzu's Edman sequencing system to provide researchers and scientific customers withN-terminal protein sequencing based on Edman degradationservices. Using our sequencing system, the sequence information of 30 amino acids at the N-terminal can be determined. With a specific protein loading system, 60-70 amino acids at the N-terminal can be determined. Bio-Techne has also established a platform for N-terminal sequencing using advanced LC-MS/MS technology, capable of detecting closed and modified protein termini, complementingN-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
Protein full sequence determination
How to order?
