Workflow of De Novo Antibody Sequencing

Antibody de novo sequencing is an innovative technology that enables scientists to identify and characterize the amino acid sequences of unknown antibodies. This technique is of great significance in biomedical research and drug development.

 

Sample Preparation

The first step in antibody de novo sequencing is sample preparation. This involves extracting antibodies from biological samples and purifying them to remove impurities. Common purification methods include affinity chromatography, ion-exchange chromatography, and gel filtration chromatography. These methods efficiently separate antibodies, providing high-purity samples for subsequent analysis.

 

Antibody Digestion

After obtaining purified antibody samples, the next step is to digest the antibodies into smaller peptides. This is typically achieved through enzymatic digestion, with trypsin being the most commonly used enzyme. Trypsin specifically cleaves antibodies at designated sites, producing peptides suitable for mass spectrometry analysis.

 

Peptide Separation and Purification

Following enzymatic digestion, the generated peptides need further separation and purification. This step is usually performed using liquid chromatography (LC). Liquid chromatography separates different peptides based on their physicochemical properties, ensuring each peptide can be accurately detected and analyzed.

 

Mass Spectrometry Analysis

Mass spectrometry (MS) analysis is the core step in antibody de novo sequencing. In this step, the separated peptides are introduced into a mass spectrometer, where their mass-to-charge ratio (m/z) is measured. Common mass spectrometry techniques include tandem mass spectrometry (MS/MS) and high-resolution mass spectrometry (HR-MS). MS analysis provides precise mass information of the peptides, laying the foundation for subsequent sequence reconstruction.

 

Data Analysis and Sequence Reconstruction

The data generated from mass spectrometry need to be processed and analyzed using bioinformatics tools. Commonly used software includes Mascot, MaxQuant, and Proteome Discoverer. These tools match peptide sequences based on mass spectrometry data and ultimately reconstruct the complete amino acid sequence of the antibody. The data analysis process considers factors such as mass spectrum peak intensity, fragment ion spectra, and peptide physicochemical properties.

 

Result Validation

To ensure the accuracy of antibody de novo sequencing, result validation is usually required. This can be achieved by comparing the obtained sequences with known antibody sequences. Additionally, synthetic peptides can be generated and validated through mass spectrometry or biological functional assays to verify the correctness and functionality of the sequenced antibodies.

 

Antibody de novo sequencing is a complex and precise technology. Its workflow includes sample preparation, antibody digestion, peptide separation and purification, mass spectrometry analysis, data analysis and sequence reconstruction, and result validation. Through these steps, scientists can accurately identify and characterize the amino acid sequences of unknown antibodies, providing crucial support for antibody research and drug development. As technology continues to advance, antibody de novo sequencing will play an increasingly important role in biomedical research.