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    Analysis of the N-Terminal Sequence of a Protein

      Protein synthesis starts from the N-terminus and the composition of the N-terminal sequence of the protein affects the overall biological function of the protein. For example, the N-terminal sequence affects the half-life of the protein and is related to the location of the protein subcellular organelle. Analysis of protein N-terminal sequencing helps in the analysis of protein's higher structure and reveals the biological function of the protein. With the development of modern pharmaceutical industry, a large number of protein and peptide drug molecules have emerged. The analysis and confirmation of the N-terminal sequence of these protein drug molecules is also an important part of the quality control in the pharmaceutical industry. The N-terminal region is also an important structure and functional site for proteins and peptides. Most proteins can be identified by a few amino acid residues at the N-terminus. For example, the identification of the N-terminal artificial modification sites of protein and peptide drugs, such as cyclization modification and methylation modification, can lay the foundation for improving their degradation stability and prolonging the efficacy.

       

      Protein N-terminal Sequencing Techniques

      1. Edman Degradation

      (1) Principle of Edman Degradation Sequencing

      Edman degradation is a very mature and classical method of protein and peptide N-terminal sequencing, widely used in the biotechnology field. The principle of Edman degradation sequencing is mainly to identify the type of amino acids one by one from the N-terminal of the protein through cyclic reactions, thereby determining the N-terminal sequence of the protein. Phenyl isothiocyanate (PITC) reacts with the N-terminal amine of the peptide to be analyzed under alkaline conditions to form a phenylaminothioamide derivative, and then the coupling product is treated with acid. The N-terminus of the peptide chain is selectively cleaved, releasing the thiazolinone phenylamine derivative of this amino acid residue. The extracted amino acid derivative is converted into stable ethyl carbamide-sulfonyl amino acid (PTH-amino acid) under strong acid conditions. The type of degraded PTH amino acid can be analyzed by HPLC or electrophoresis to obtain the N-terminal sequence information of the protein or peptide.

      (2) Advantages of Edman Degradation Sequencing

      The Edman degradation method has been widely used as the gold standard for detecting the N-terminal sequence of existing protein samples. It is a valuable research tool and the most reliable sequencing method for the entire purified protein N-terminal sequence analysis.

      (3)Disadvantages of Edman Degradation Sequencing

      The Edman degradation method is subject to many limitations, such as the protein or peptide used for sequence analysis must be of high purity, not suitable for high-throughput analysis, and the sensitivity is not enough.

       

      Mass Spectrometry

      The protein N-terminal sequencing technique based on mass spectrometry can simultaneously determine multiple protein N-terminal sequences at one time, especially the Electrospray Ionization (ESI) and Matrix-Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF). The high sensitivity, high accuracy, high resolution, and high throughput of biological mass spectrometry technology provide important tools for protein N-terminal sequencing. The N-terminal sequencing technique based on mass spectrometry can achieve sequence determination of N-terminal closure and PEGylated proteins, which is complementary to Edman sequencing.

       

      Chemical Labeling Combined With Mass Spectrometry Analysis

      Many research methods for N-terminal peptides use mass spectrometry technology in combination with various chemical methods and bioenzyme methods. For example, proteins are capped by reducing, alkylating and guanidinating the side chain amines. The free N-terminus is labeled with different biotin reagents. The labeled proteins are digested with trypsin, and the labeled N-terminal peptides are separated by an avidin affinity system. The sequence of the N-terminal peptide is obtained by de novo sequencing with MALDI-TOF/MALDI-TOF-PSD MS.

       

      With the continuous development and improvement of classical methods, various chemical modifications based on mass spectrometry, as well as enzyme-assisted techniques, protein N-terminal sequencing has obtained rich sequence information, providing a strong basis for accelerating the identification of terminal peptides. The N-terminal sequencing analysis technique for thousands of proteins in complex biological systems is still a major challenge we face. In particular, more targeted research strategies are needed for large-scale and more detailed determination of N-terminal modification diversity.

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