The key to Edman sequencing is the reagent phenylisothiocyanate (PITC), which is called the Edman reagent. When the N-terminus of a protein is not blocked, PITC easily interacts with the N-terminal α residue of a peptide at an alkaline pH, thereby producing a phenylthiocarbamoyl amino acid derivative. Without destroying the peptide bonds of other residues in the sequenced peptide, the N-terminal sequence can be accurately determined by removing and identifying the labeled N-terminal amino acid. When the N-terminus of a protein is cyclized and blocked, the α-amino group at the N-terminus is modified (α-amino acetylation, methylation, pyroglutamate, etc.), resulting in a lack of free α-amino groups at the N-terminus, which results in the inability of PITC to actually bind to the protein, and ultimately makes it impossible for the Edman degradation reaction to proceed directly. Therefore, Edman sequencing has certain limitations.
If the N-terminus has been chemically modified or non-α-amino acids are encountered, Edman degradation sequencing cannot be used. For N-terminal blocking, samples that meet the requirements can be selected according to the specific situation (50% of natural protein N-termini have been modified in nature, and common modifications include acetylation, methylation, pyroglutamate, etc.); if the N-terminus is blocked due to the reaction of detergents or chemicals in the solution with the functional groups of the N-terminus of the protein sample, or the pH value of the solvent used for separation and purification is too high, select the corresponding enzyme to remove the modification of the N-terminus of the protein, or use mass spectrometry to identify the modification of the N-terminus; if the modification that causes the protein N-blocking is known, the corresponding protease can be used to remove the modification of the N-terminus, and then the Edman degradation reaction can be used for sequencing.
The current Edman sequencing technology can only measure the 30-60 amino acid residue sequence of the N-terminus of the protein, which limits the analysis and determination of the full-length protein sequence. Of course, Edman sequencing can also be used to analyze the full-length protein sequence by pre-treating the protein sample. First, the protein is cut into multiple short peptides using a protease, and then the short peptide sequences are measured in sequence using the Edman sequencing method. Finally, the full-length protein sequence can be measured by sequence splicing.
Edman sequencing is usually not used to determine the location of disulfide bonds, and the throughput is low, so it is impossible to analyze multiple proteins simultaneously. The advantages of mass spectrometry identification and Edman sequencing can be combined to achieve high-throughput protein identification and accurate sequence determination.