Mechanism of De Novo Antibody Sequencing

Antibody de novo sequencing (ADNS) is a method used to determine the amino acid sequence of antibodies, which plays a critical role in biomedical research and therapeutic antibody development. Given the widespread application of monoclonal antibodies (mAbs) in treating cancer, infectious diseases, and autoimmune disorders, accurately resolving antibody sequences is crucial for ensuring their efficacy and safety.

 

The core principle of ADNS is the analysis of antibody peptides using mass spectrometry (MS) to deduce the complete amino acid sequence of the antibody. MS technology can efficiently and accurately determine the molecular mass of peptides, and when combined with bioinformatics tools, enables de novo sequencing of antibodies. Unlike traditional genetic sequencing methods, MS does not require prior genetic information and directly analyzes the antibody protein, hence the term "de novo" sequencing.

 

Technical Steps

1. Antibody Sample Preparation

The initial step in ADNS involves preparing the antibody sample. The sample preparation steps include antibody purification, reduction, alkylation, and enzymatic digestion. The detailed steps are as follows:

 

(1) Antibody Purification: Antibodies are isolated and purified from mixed samples using protein A/G affinity chromatography.

(2) Antibody Reduction: Disulfide bonds in the antibody are cleaved using reducing agents such as dithiothreitol (DTT), separating the heavy and light chains.

(3) Alkylation: Free thiol groups are alkylated using iodoacetamide (IAM) to prevent reformation of disulfide bonds.

(4) Enzymatic Digestion: The antibody is digested into smaller peptides using specific proteases like trypsin and chymotrypsin.

 

2. Mass Spectrometry Analysis

The prepared peptide samples are analyzed using liquid chromatography-mass spectrometry (LC-MS). Liquid chromatography separates the peptides, and the mass spectrometer detects each peptide, generating a mass spectrum (MS spectrum).

 

3. Data Analysis

After obtaining the mass spectrum, bioinformatics software is used to analyze the MS data. The main steps of the analysis include:

 

(1) Peptide Matching: Peptide sequences are initially identified by matching the m/z (mass-to-charge ratio) data from the mass spectrum with known peptide masses.

(2) Sequence Assembly: The identified peptide sequences are assembled to deduce the complete amino acid sequence of the antibody.

(3) Result Validation: The accuracy of the results is verified through repeated experiments and additional techniques such as tandem mass spectrometry (MS/MS).

 

Applications and Prospects

ADNS has broad applications in basic research, drug development, and clinical practice. Specific applications include:

 

1. Antibody Characterization and Optimization

Researchers can obtain detailed information about the antibody’s amino acid sequence and structural conformation through ADNS, enabling the optimization of antibody affinity and specificity.

 

2. Therapeutic Antibody Development

ADNS aids in identifying and validating candidate antibody sequences during drug development, ensuring their consistency and efficacy.

 

3. Biologics Analysis

Quality control and consistency evaluation of marketed antibody drugs to ensure product safety and effectiveness.

 

In conclusion, ADNS provides essential technical support for antibody research and application. With continuous advancements in MS technology and bioinformatics tools, the efficiency and accuracy of ADNS will further improve, driving innovation in the biomedical field.