The Basic Process of Antibody Sequencing

Antibody sequencing is a technique used to determine the amino acid sequence of specific antibody molecules in monoclonal or polyclonal antibodies. This provides key information about the specificity, activity, structure, and affinity of the antibody with the antigen, which is critical for antibody engineering, vaccine development, disease diagnosis, and treatment research. Mass spectrometry is the gold standard for antibody sequencing, and in this issue, we mainly introduce the basic process of antibody sequencing based on mass spectrometry.

 

Sample Preparation

Initially, a sample containing the target antibody needs to be prepared. For monoclonal antibodies, this usually involves extracting antibodies from hybridoma cell culture. For polyclonal antibodies, it may require extraction from serum or other biological fluids.

 

Antibody Purification

Antibody samples usually need to be purified through Protein A, Protein G, or specific affinity columns to remove non-specific proteins and other impurities.

 

Lysis and Reduction

The purified antibody is lysed into smaller fragments, generally by treatment with reducing agents to break disulfide bonds, yielding separate light and heavy chains.

 

Trypsin Digestion

The lysed antibody is treated with trypsin or other specific proteases to produce smaller peptides that can undergo sequence analysis.

 

Peptide Purification and Separation

The peptides obtained are further purified and separated by high-performance liquid chromatography (HPLC) or similar techniques.

 

Mass Spectrometry Analysis

Purified peptides are analyzed using mass spectrometry techniques (such as MALDI-TOF or electrospray tandem mass spectrometry (ESI-MS/MS)) to determine their mass and sequence.

 

Sequence Assembly and Analysis

Using bioinformatics tools and databases, peptide sequence information obtained from mass spectral data is assembled into complete antibody light and heavy chain sequences. This may involve comparing with known sequence databases to identify and annotate sequences.

 

It should be noted that the target of sequencing can be the totality of antibodies (i.e., all antibodies obtained from multiple B cells) or a single clone of antibodies. Additionally, the choice of techniques and tools may vary depending on the specific research purpose and conditions.