The main contents of general chemical drug substance structure confirmation research include plane structure, three-dimensional structure, crystal form and crystal water/crystal solvent. For drug molecules of custom peptide synthesis , it is more necessary to conduct structural confirmation research in amino acid composition analysis, amino acid sequence analysis, disulfide bond analysis, etc.

1. Molecular weight determination

Like chemical raw materials, the structural confirmation of synthetic peptide drugs also includes the determination of molecular weight. Generally, mass spectrometry is used to determine the monoisotopic mass, and the mass deviation should be within ±1.0 mass units of the theoretical value; for molecular weights of 2 kDa For the above peptides, it is more suitable to use a high-resolution mass spectrometer

2. Analysis of amino acid composition

Amino acid composition analysis can indicate whether the composition of the peptide is correct, not only to prove the structure of the peptide, but also to reflect the purity of the sample (whether it contains synthetic impurities) to a certain extent. In the analysis of amino acid composition, attention should be paid to the chromatographic behavior of various amino acids involved, especially unnatural amino acids and amino acid derivatives, so as to accurately conduct qualitative and quantitative analysis. In addition, a suitable hydrolysis method should be established in this analysis, because different hydrolysis conditions may have a greater impact on the recovery of certain amino acids, such as glutamine at pH<3

(Gln) will be hydrolyzed by acid-catalyzed deamination; while at pH>7, Gln is not easy to form a six-membered ring and is more stable

3. Amino acid sequence analysis

As with other molecules, elucidation of chemical composition is the basis for determining peptide identity, including elucidation of amino acids and their order in the peptide chain, i.e. 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, which can be used to elucidate complex peptides containing disulfide bonds. Peptide sequence determination by tandem mass spectrometry for the primary structure of calcined salmon typically uses collision-induced dissociation (CID) fragmentation, but if sequence coverage is poor, other mass spectrometry fragmentation techniques should be considered. For long peptides containing more than 20 amino acid residues, if the sequence cannot be directly determined, the analysis of the peptide map can be carried out. The peptides were hydrolyzed, and each peptide fragment was identified by liquid chromatography-mass spectrometry (LC-MS).

4. Disulfide bond analysis

The correct number and position of disulfide bonds are the structural guarantee for the biological activity of polypeptides and proteins. In the process of researching synthetic or natural polypeptide products, the analysis of disulfide bonds is one of the important contents to be investigated. If a synthetic polypeptide drug molecule contains a disulfide bond, it is generally necessary to

These drugs are reduced by reducing reagents such as dithiothiol (DTE), dithiothreitol (DTT), or tris(2-carboxyethyl)phosphine (TCEP), followed by mass spectrometry analysis. The difference in mass can determine the number of disulfide bonds it contains. Among them, for polypeptides containing multiple pairs of disulfide bonds, the localization analysis of the disulfide bonds should also be performed. Partial reduction is a widely accepted method for localizing disulfide bonds in which peptides are digested under controlled conditions so that disulfide bonds with different reduction kinetics are reduced and separated by tandem mass spectrometry. and analysis to complete the positioning of the disulfide bond.

5. Secondary structure analysis

Peptides containing more than 40 amino acids typically exhibit a high degree of conformational flexibility, characterized primarily by random helices and some degree of secondary structure (eg, alpha helices and/or beta sheets). The order of the structure may have an important effect on the biological activity of some peptides, so the elucidation of secondary structure is also part of the structural characterization of peptides. Commonly used methods such as circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), X-ray crystallography, Raman spectroscopy and fluorescence spectroscopy are used to analyze the secondary structure of polypeptide drugs.

Attachment:

Mass spectrometry fragmentation technology content:

low energy CID

High energy CID

Scope of application

Shorter amino acid sequence peptides with 2 or 3 charges predominately

High Energy Collision Dissociation (HCD)

Widely used in the study of protein post-translational modifications

Based on Electron Induced Electron Capture Dissociation (ECD)

Peptides with longer amino acid sequences and mainly with 3 or more charges

Electron Transfer Dissociation (ETD)

Widely used in the study of protein post-translational modifications

Peptides with longer amino acid sequences and mainly with 3 or more charges