Why can the Edman method only determine protein sequences of about 50 amino acids or less?
Proteins are crucial molecules for maintaining normal physiological activities in living organisms. They determine the structure and activity of cells, provide mechanisms for signal transmission between cells and tissues, and catalyze chemical reactions that support metabolism. The structure of a protein determines its function (or dysfunction).
The Edman degradation method is a primary technique for determining protein sequences. In this process, phenyl isothiocyanate (PITC) reacts with the N-terminal free α-residue to form a phenylthiocarbamoyl (PTH) derivative, which is then cleaved to produce a thiazolinone derivative (ATZ) and a new N-terminal. The released phenylthiohydantoin amino acid (PTH-amino acid) is stable and can be identified using electrophoresis or high-performance liquid chromatography. After the first amino terminal residue is liberated, the same reaction cycle is performed on the remaining polypeptide chain to release the next residue, and repeating this cycle can reveal the complete sequence information of the polypeptide.
Edman degradation can identify amino acids one by one through chemical reactions, but the method is slow (one cycle takes 1 hour). Through cyclic derivatization, it is limited to reliably determining sequences of about 30 amino acid residues in a peptide chain, with a maximum of 50-60 amino acid residues.
Some techniques can further improve the detection limits on this basis, but this manipulation can lose some overall protein information and greatly reduce detection accuracy, making it impossible to distinguish many close mass signals. If the sample purity meets the requirements and the protein N-terminal has a free α-residue, it requires about 100 pmol of pure peptide sample, with each amino acid identification efficiency >99%. If Edman degradation is performed when there is no free α-residue at the N-terminal (N-terminal is blocked or chemically modified), it cannot clearly identify the detection signal, so it cannot be used to accurately detect and identify post-translationally modified proteins.
Biotech company Baite Park uses Shimadzu's Edman sequencing system to provide researchers and scientific customers withN-terminal protein sequencing based on Edman degradationservices. By using our sequencing system, the N-terminal sequence information of 30 amino acids can be determined. Using a specific protein loading system, the N-terminal 60-70 amino acids can be determined. Baite Park Biotech has also established a platform for N-terminal sequencing with advanced LC-MS/MS technology, which can determine 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
Full protein sequence determination
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