Mass Spectrometry Analysis of Disulfide Bond Mismatch
Disulfide bonds are important structural elements in proteins, playing a crucial role in determining the three-dimensional structure and function of proteins. However, during the development of biopharmaceuticals, mispairing of disulfide bonds can lead to structural changes and loss of function in proteins, making the analysis of disulfide bonds particularly important. Mass spectrometry is an efficient and accurate method for analyzing disulfide bonds, capable of performing disulfide bond pairing analysis at the protein level. It can determine both the location and pairing mode of disulfide bonds. The specific steps are as follows.
1. Sample Preparation
First, treat the protein sample to break non-disulfide linkages, then use reducing agents such as DTT or β-mercaptoethanol to reduce disulfide bonds to thiols.
2. Thiol Protection
React thiols with protecting agents such as IAM or NEM to prevent reformation of disulfide bonds.
3. Enzymatic Digestion
Use specific enzymes such as trypsin or glucosidase to cleave the protein and obtain disulfide bond-containing peptides.
4. Mass Spectrometry Analysis
Conduct LC-MS/MS analysis on the obtained peptides. By analyzing the mass and mass distribution of the peptides, the locations and pairing modes of disulfide bonds can be determined.
5. Data Processing
Process mass spectrometry data using software or databases to obtain precise disulfide bond pairing information.
The above are the basic steps for disulfide bond mispairing analysis using mass spectrometry. It is worth noting that although this method is accurate, it requires high sample quality and operational skills, thus experienced experimenters are needed.
BiotechPack, A Biopharmaceutical Characterization and Multi-Omics Mass Spectrometry (MS) Services Provider
Related Services:
Protein Mass Spectrometry Identification
Post-Translational Modification Quantitative Proteomics Analysis
How to order?
