Limitations of Edman Sequencing
The most accurate method for identifying proteins is to use protein sequencing methods. The two main methods of protein sequencing are the Edman degradation method and mass spectrometry. Edman sequencing complements mass spectrometry and is superior in sequencing animal peptides (where no sequence database is available) compared to mass spectrometry.
The key to Edman sequencing is the reagent phenyl isothiocyanate (PITC), known as the Edman reagent. In the absence of N-terminal blockage, PITC easily reacts with the N-terminal α-residue of the peptide under basic pH, producing phenylthiocarbamoyl amino acid derivatives. The marked N-terminal amino acid can be removed and identified without destroying the peptide bonds of other residues in the sequenced peptide, allowing for accurate determination of the N-terminal sequence. When the protein's N-terminus undergoes cyclization or blockage, the N-terminal α-amino is modified (such as acetylation, methylation, pyroglutamylation, etc.), making the N-terminus lack a free α-amino group, preventing PITC from binding to the protein and ultimately hindering the progression of the Edman degradation reaction. Thus, Edman sequencing has certain limitations.
If the N-terminus is chemically modified or contains non-α-amino acids, the Edman degradation method cannot be used for sequencing. For blocked N-termini, samples that meet the requirements can be selected (50% of natural proteins in nature have modified N-termini, with common modifications including acetylation, methylation, pyroglutamylation, etc.). If the blockage is due to detergents or chemicals in the solution reacting with the protein sample's N-terminal functional groups, or if the solvent used for separation and purification has a high pH, appropriate enzymes can be used to remove the modifications, or mass spectrometry can be used to identify the N-terminal modifications. If the modification causing the protein N-terminal blockage is known, corresponding proteases can be used to remove the modifications, allowing Edman degradation to proceed for sequencing.
Current Edman sequencing technology can only determine the sequence of 30-60 amino acid residues at the protein's N-terminus, which limits the analysis and determination of the full-length protein sequence. However, by pre-treating the protein sample, Edman sequencing can be used to analyze the full-length protein sequence. First, proteases are used to cleave the protein into multiple short peptides, then Edman sequencing is used to sequentially determine the short peptide sequences. Finally, the sequences are assembled to determine the full-length protein sequence.
Edman sequencing is typically unable to determine the positions of disulfide bonds and has low throughput, making it difficult to analyze multiple proteins simultaneously. By combining the advantages of mass spectrometry and Edman sequencing, high-throughput protein identification and precise sequence determination can be achieved.
Biotech company Biotai Park uses Shimadzu's Edman sequencing system to provide researchers and scientific customers withN-terminal protein sequencing based on Edman degradationservices. Our sequencing system can determine the sequence information of the first 30 amino acids at the N-terminus. Using a specific protein loading system, it can determine 60-70 amino acids at the N-terminus. Biotai Park has also established a platform for N-terminal sequencing using advanced LC-MS/MS technology, which can identify closed and modified protein termini, complementingN-terminal protein sequencing based on Edman degradationto ensure smooth N-terminal sequencing services.
Related services:
Protein N/C-terminal sequencing
Biopharmaceutical N/C-terminal sequencing
Full protein sequence determination
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