Antibody Amino Acid Sequencing Basic Process

The sequencing of antibody amino acids is carried out to determine the amino acid sequence of the antibody heavy chain and light chain. This is usually done to address issues of antibody specificity, activity, structure, and affinity for the antigen. The sequencing of antibody amino acids mainly includes the following steps.

 

Antibody Preparation and Purification

First, the target antibody needs to be obtained from the corresponding cell culture or animal model. This step may involve the use of specific cell lines (such as hybridoma cells) to produce monoclonal antibodies, or the acquisition of polyclonal antibodies from immunized animals. Afterwards, high-quality antibodies are purified from complex samples using affinity chromatography or other purification techniques.

 

Antibody Cleavage

Antibodies are composed of two types of polypeptide chains: light chains and heavy chains. In order to carry out amino acid sequence analysis, reducing agents (such as dithiothreitol or β-mercaptoethanol) are usually used to cleave the disulfide bonds in the antibody molecule and separate these chains.

 

Enzymatic Digestion

Next, specific proteases (such as trypsin or chymotrypsin) are used to cut the purified and cleaved antibody polypeptide chains, producing smaller peptide segments. This step is to generate peptide segment sizes suitable for mass spectrometry analysis.

 

Peptide Purification

After enzymatic digestion, the peptide mixture may need to be further purified and separated for subsequent mass spectrometry analysis. Liquid chromatography (LC) is a commonly used purification method that can effectively separate peptide segments based on their physicochemical properties (such as hydrophilicity or hydrophobicity).

 

Mass Spectrometry Analysis

The purified peptides are analyzed by mass spectrometry, with common technologies including electrospray ionization (ESI) mass spectrometry and matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Mass spectrometry analysis can provide accurate mass and sequence information for peptide segments.

 

Sequence Identification

Using mass spectrometry data, in conjunction with bioinformatics tools and databases, the amino acid sequence of the peptide segments can be identified. This usually involves matching experimental data with theoretical peptide masses and sequences in known protein databases to identify and determine the precise amino acid sequence of the antibody.

 

Sequence Analysis and Verification

Finally, by analyzing the obtained amino acid sequence, the variable (V) regions and constant (C) regions of the antibody can be identified, as well as determining the subtype of the antibody and potential post-translational modifications. Further experiments, such as protein sequence alignment and functional verification, may be required to confirm the accuracy and biological significance of the sequence.

 

The above is just a comprehensive overview, and the actual sequencing of antibody amino acids may involve more steps and details, and will be adjusted according to the equipment and technology used.