The synthesis of proteins initiates at the N-terminus, and the sequence composition of the N-terminus plays a crucial role in determining the overall biological function of the protein. For instance, the N-terminus sequence affects the protein's half-life and influences the subcellular organelle location. Analyzing the N-terminal sequence of proteins through N-terminal sequencing can help reveal the high-level structure and biological functions of proteins. The emergence of numerous protein and peptide drug molecules in the modern pharmaceutical industry has made it essential to analyze and confirm their N-terminal sequence as a vital aspect of quality control. The ICH Q6B guidelines require protein drug manufacturers to provide information on the N-terminal region. Moreover, the N-terminal region is an essential structural and functional site of proteins and peptides, and most proteins can be identified by the few amino acid residues at the N-terminus. For example, identifying artificial modification sites on the N-terminus of protein and peptide drugs, such as cyclization and methylation, can enhance their degradation stability and prolong their efficacy.
N-Terminal Sequencing Techniques for Proteins and Peptides
Edman degradation
The principle of Edman degradation sequencing
Edman degradation sequencing is a classic and mature method for determining the N-terminal sequence of proteins and peptides, commonly used in biotechnology. The method involves identifying the amino acid types one by one from the N-terminus of the protein through a cycle reaction to determine the N-terminal sequence. The N-terminal amino group of the peptide to be analyzed reacts with phenyl isothiocyanate (PITC) under alkaline conditions to form aniline thioformamide derivative. The coupled product is then treated with acid to selectively cleave the N-terminus of the peptide chain, releasing the thiazolinone aniline derivative of the amino acid residue. The extracted amino acid derivatives are converted into stable hydantoin thiourea amino acids (PTH-amino acids) under strong acidic conditions. The degraded PTH-amino acid species can be analyzed using HPLC or electrophoresis to obtain N-terminal sequence information on proteins or peptides.
The advantages of Edman degradation sequencing
Edman degradation method is the gold standard for N-terminal sequence testing of existing protein samples, making it a valuable research tool for N-terminal sequence analysis of the entire purified protein and the most reliable sequencing method.
The disadvantages of Edman degradation sequencing
The Edman degradation method is subject to several restrictions, such as the requirement for high-purity protein or peptide for sequence analysis. It is not suitable for high-throughput analysis, and the sensitivity is not sufficient.
Mass spectrometry
Mass spectrometry-based protein N-terminal sequencing technology can determine the protein N-terminal sequence at once, especially with the electrospray ionization (ESI) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF). Mass spectrometry's application in protein structure analysis has undergone a revolutionary leap, with high-sensitivity, high-accuracy, high-resolution, and high-throughput biological mass spectrometry technologies providing an important choice for protein N-terminal sequencing. N-terminal sequencing technology based on mass spectrometry can determine the sequence of N-terminal blocking and PEGylated protein, which is complementary to Edman sequencing. This analysis can confirm and identify the high-level structural integrity of recombinant protein drugs and the modification site of the recombinant protein's N-terminal sequence. Therefore, N-terminal sequence sequencing can lay the foundation for comparing the original antibody's sequence and modification.
Chemical labeling combined with mass spectrometry
Many research methods for N-terminal peptides use a combination of mass spectrometry technology and a variety of chemical methods and biological enzymatic methods. For example, the protein is blocked by reduction, alkylation and guanidylation of side chain amino groups. The free N-terminal is labeled with different biotin reagents. After the labeled protein is digested with trypsin, the labeled N-terminal peptide is separated by the avidin affinity system, and then passed through MALDI-TOF / MALDI-TOF- PSD MS de novo sequencing to obtain the sequence of N-terminal peptide.
Future development of N-terminal sequencing
With the continuous development and improvement of classical methods, various chemical modifications based on mass spectrometry, and enzymatic assisted technologies, N-terminal sequence analysis of proteins and peptides has obtained rich terminal peptide sequence information, which provides a powerful basis and accelerate the identification of high-level structures of protein drugs and the study of modification sites. The N-terminal sequencing analysis technology of thousands of proteins in complex biological systems is still a huge challenge we face, especially for a more detailed determination of the N-terminal modification diversity on a large scale, a more targeted research strategy is also needed. The future development direction may be the exploration of chemical modification reagents and modification conditions with better selectivity and more controllable modification.
Sign in to leave a comment.