Principle of De Novo Antibody Sequencing

Antibodies are essential molecules in the immune system that specifically bind to antigens, marking foreign substances for clearance by the immune system. With the extensive application of antibodies in both basic research and clinical settings, understanding their sequences and characteristics has become crucial. Antibody de novo sequencing is a technique used to determine antibody sequences without a reference sequence, directly obtaining the amino acid sequence of the antibody through mass spectrometry.

 

Principle of Antibody De Novo Sequencing

1. Fundamentals of Mass Spectrometry

The core technology underlying antibody de novo sequencing is mass spectrometry (MS). MS is a technique for analyzing the composition of substances by measuring the mass-to-charge ratio (m/z) of ions. A mass spectrometer comprises three main components: an ion source, a mass analyzer, and a detector. The sample is ionized into charged ions in the ion source, these ions are then separated according to their mass-to-charge ratio in the mass analyzer, and finally detected and recorded by the detector.

 

2. Antibody Sample Preparation

Antibody de novo sequencing requires the enzymatic digestion of the antibody sample, typically using proteases such as trypsin to cleave the antibody into smaller peptide fragments. These peptide fragments can be resolved in mass spectrometry analysis and are more amenable to sequencing.

 

3. Acquisition of Mass Spectrometry Data

The digested peptide fragments are introduced into the mass spectrometer, where they undergo electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) to generate charged ions. These ions are directed into the mass analyzer, separated based on their mass-to-charge ratio, and recorded by the detector. The resulting ion signals are analyzed to produce mass spectra of the peptide fragments.

 

4. Sequence Reconstruction

Peaks in the mass spectrum represent the mass-to-charge ratios of peptide fragments. By analyzing the positions and intensities of these peaks, the amino acid sequences of the peptides can be inferred. Tandem mass spectrometry (MS/MS) can be employed to further fragment the peptide fragments through two stages of mass spectrometry analysis, providing more detailed structural information. This information is integrated to reconstruct the complete amino acid sequence of the antibody.

 

5. Data Analysis and Validation

Mass spectrometry data requires complex computational analysis using specialized software and algorithms for sequence inference and alignment. To ensure accuracy, multiple replicate experiments are typically conducted, and different proteases are used for digestion to obtain a variety of peptide fragments, further validating the sequence accuracy.

 

Antibody de novo sequencing is a powerful technique that can directly determine the amino acid sequence of antibodies without a reference sequence. By utilizing mass spectrometry and advanced data analysis methods, researchers can gain in-depth insights into the structure and function of antibodies, providing critical support for the basic research and clinical applications of antibodies.