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Application of tandem mass spectrometry in structural confirmation of synthetic peptide drugs (1)
Peptide drugs are a kind of important drug molecules between macromolecular protein/antibody drugs and small molecule drugs. They are widely used in the field of disease treatment because of their high biological activity, high targeting specificity, high selectivity, low toxicity and other advantages. In recent years, the in-depth application of microspheres, liposomes, polyethylene glycol (PEG) modification and other methods has solved the problems of poor stability of peptide drugs, easy degradation in vivo, short half-life and other problems of poor drug properties, and promoted the development and utilization of peptide drugs
A number of preclinical experiments and clinical studies have shown that polypeptide drugs have a wide range of effects and can be used to treat osteoporosis, diabetes, prostate disease, endometriosis, acromegaly, ulcer, hypothyroidism and other diseases. The future commercial value and development prospects are huge. So far, the FDA of the United States has approved more than 90 active peptide drugs to enter the market. Many of the approved peptides are synthesized by chemical methods, including liquid phase synthesis or solid phase synthesis, and more synthetic peptide drugs are under pre-marketing research.
With the continuous growth of the number of research and development of synthetic peptide drugs, the research on the structure confirmation of such drugs is also being carried out. At present, the commonly used N-terminal sequencing method of peptide and protein is Edman degradation method, but it has some disadvantages such as unclear results and low sensitivity. For example, amino acid residues such as tryptophan and cysteine may not be detected due to background interference, so Edman degradation method is difficult to characterize trace peptides, N-terminal blocking peptides or polypeptide mixtures. With the development of sequencing technology and instruments, tandem mass spectrometry has become a highly efficient, highly sensitive and high-throughput peptide sequencing tool, playing an important role in the molecular structure confirmation of synthetic peptide drugs.
Research content of structural confirmation of synthetic peptide drugs
The main contents of the research on the structure confirmation of general chemical raw materials include plane structure, three-dimensional structure, crystal form and crystal water/crystal solvent. To synthesize polypeptide drug molecules, it is more necessary to carry out structural confirmation research in amino acid composition analysis, amino acid sequence analysis, disulfide bond analysis and other aspects.
Molecular weight determination
Like chemical raw materials, the determination of molecular weight is also included in the structural confirmation of synthetic peptide drugs, and the determination of single isotope mass is generally carried out by mass spectrometry
Amino acid composition analysis
Amino acid composition analysis can show whether the composition of polypeptide is correct, which can not only prove the structure of polypeptide, but also reflect the purity of sample to a certain extent (whether it contains synthetic impurities (exenatide impurities). When analyzing the composition of amino acids, pay attention to the chromatographic behavior of all kinds of amino acids involved, especially non-natural amino acids and amino acid derivatives, so as to accurately conduct qualitative and quantitative analysis
Amino acid sequence analysis
Like other molecules, the elucidation of chemical components is the basis for determining the identity of peptides, including elucidation of amino acids and their sequence in the peptide chain, i.e. the primary structure/sequence. The traditional amino acid sequencing method is Edman degradation method, but this method is generally used to determine N-terminal amino acids; In addition, the combination of Edman sequencing and tandem mass spectrometry is also a commonly used method at present, which can be used to clarify the primary structure of complex peptides containing disulfide bonds, such as calcitonin salmon. Collision-induced dissociation (CID) fragmentation technology is usually used to determine peptide sequence by tandem mass spectrometry, but if the sequence coverage is poor, other mass spectrometry fragmentation technology should be considered
Disulfide bond analysis
The correct number and position of disulfide bonds is the structural guarantee for the biological activity of peptides and proteins. In the process of research on synthetic or natural peptide products, the analysis of disulfide bonds is one of the important contents to be investigated. If there are disulfide bonds in the molecule of synthetic polypeptide drugs, the reduction reagent such as dithiomercaptan (DTE), dithiothreitol (DTT) or tris (2-carboxyethyl) phosphine (TCEP) is generally used to reduce such drugs, and then mass spectrometry analysis is carried out. The number of disulfide bonds can be determined by comparing the quality difference before and after reduction. Among them, the location analysis of disulfide bonds should also be carried out for peptides containing multiple pairs of disulfide bonds. Partial reduction is a widely accepted method for locating disulfide bonds. In this method, polypeptides are digested under controlled conditions, so that disulfide bonds with different reduction kinetics are reduced. After separation and analysis by tandem mass spectrometry, disulfide bonds can be located
Secondary structure analysis
Peptides containing more than 40 amino acids usually exhibit a high degree of conformational flexibility, characterized by random helices and a certain degree of secondary structure (e.g α Spiral and/or β Fold). The order of the structure may have an important impact on the biological activity of some peptides, so the elucidation of the secondary structure is also a part of the structural characterization of polypeptides. Circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), X-ray crystallography, Raman spectroscopy and fluorescence spectroscopy are commonly used to analyze the secondary structure of polypeptide drugs
Application of tandem mass spectrometry in structural confirmation of synthetic peptide drugs
Tandem mass spectrometry is the combination of more than two levels of mass analysis in time or space, which is used to determine the mass relationship between the precursor ion (Pre-cursorion) in the first level of mass analyzer and the product ion (Product-ticm) in the second level of mass analyzer.
Article from custom peptide synthesis company in China -Omizzur
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