Workflow of Antibody Sequencing

Antibody sequencing refers to the determination of the amino acid sequence of an antibody to understand its structure and function. This technique is crucial in antibody drug development, disease diagnosis, and basic research. The workflow of antibody sequencing includes sample preparation, antibody enrichment, protein digestion, peptide separation, mass spectrometry analysis, and data analysis. Each step is detailed below.

 

Sample Preparation

Sample preparation is the first step in antibody sequencing. Typically, purified antibodies are extracted from biological samples, ensuring no interference from other proteins. Methods such as Protein A/G column purification or immunoaffinity chromatography are used to enrich and purify the antibodies.

 

Antibody Enrichment

Antibody enrichment aims to extract the target antibody from a complex sample matrix. Common methods include Protein A/G purification and antigen affinity purification. The purpose of antibody enrichment is to improve the purity of the sample, thereby enhancing the accuracy of subsequent analyses.

 

Protein Digestion

The purified antibody needs to be digested into smaller peptides for sequencing. Trypsin is commonly used for this purpose, as it specifically cleaves the peptide chains of antibodies. The digestion reaction is carried out under appropriate conditions to ensure completeness and reproducibility.

 

Peptide Separation

After digestion, the peptides need to be separated for subsequent mass spectrometry analysis. Liquid chromatography (LC) is commonly used for separation, exploiting the peptides' hydrophilicity, hydrophobicity, and charge. High-performance liquid chromatography (HPLC) and reverse-phase liquid chromatography (RP-LC) are frequently employed methods.

 

Mass Spectrometry Analysis

Mass spectrometry (MS) analysis is the core step in antibody sequencing. Using a mass spectrometer, the molecular weight and charge ratio (m/z) of each peptide can be determined. Tandem mass spectrometry (MS/MS) is commonly used, as it further fragments the peptides and measures their fragment ions, allowing the deduction of the peptide's amino acid sequence.

 

Data Analysis

Mass spectrometry data requires detailed analysis to determine the antibody's amino acid sequence. Specialized software and databases are used to match and compare the mass spectrometry data, ultimately deriving the complete sequence of the antibody. Factors such as modifications and mutations must be considered during data analysis to ensure the accuracy and completeness of the results.

 

Through sample preparation, antibody enrichment, protein digestion, peptide separation, mass spectrometry analysis, and data analysis, detailed sequence information of antibodies can be obtained. MtoZ Biolabs provides integrate antibody sequencing service.