Analysis of Antibody Sequences Using Mass Spectrometry

Antibodies play a crucial role in biomedical research and clinical applications. Accurate antibody sequence analysis is essential for antibody engineering, drug development, and immunotherapy. In recent years, mass spectrometry (MS) has become a powerful tool for antibody sequence analysis due to its high sensitivity, high resolution, and high throughput. This article will introduce the principles, methods, and applications of mass spectrometry-based antibody sequence analysis in research and clinical settings.

 

Mass spectrometry (MS) is a technique used to identify and quantify compounds by measuring their mass-to-charge ratio (m/z). A mass spectrometer mainly consists of an ion source, a mass analyzer, and a detector. Antibody samples, after being processed by digestive enzymes, generate peptides that are ionized into gas-phase ions. These ions are separated by the mass analyzer and detected based on their mass-to-charge ratio distribution.

 

Antibody Sample Preparation and Digestion

The preparation of antibody samples is a critical step in mass spectrometry analysis. Typically, antibodies are first treated with reducing and alkylating agents to break disulfide bonds, followed by enzymatic digestion using trypsin to produce peptides. To enhance the accuracy and coverage of the analysis, additional proteases (such as chymotrypsin or glucosidase) may be employed for supplementary digestion.

 

Mass Spectrometry Data Acquisition and Processing

Mass spectrometry data acquisition includes full scan mode and data-dependent acquisition (DDA) mode. In full scan mode, all ions are detected simultaneously, which is suitable for preliminary analysis. In data-dependent acquisition mode, specific ions are selected based on pre-set ion intensity for further fragmentation (MS/MS) to obtain more detailed structural information.

 

Data processing involves peptide matching, sequence reconstruction, and database searching. Peptide matching compares experimental mass spectrometry data with theoretical data to determine peptide sequences. Sequence reconstruction infers the complete antibody sequence based on matched peptides. Database searching compares experimental data with known protein databases to identify unknown antibodies.

 

Applications of Mass Spectrometry in Antibody Sequence Analysis

1. Antibody Identification and Characterization

Mass spectrometry can be used to identify the amino acid sequence of antibodies, including the variable and constant regions of the heavy and light chains. This is crucial for functional studies and engineering modifications of antibodies.

 

2. Detection of Antibody Modifications

Mass spectrometry can accurately detect post-translational modifications (PTMs) on antibodies, such as glycosylation and phosphorylation. These modifications significantly impact antibody stability, activity, and immunogenicity.

 

3. Antibody Library Screening

In antibody drug development, mass spectrometry can be used for high-throughput screening of antibody libraries, rapidly identifying and optimizing candidate antibodies.

 

4. Clinical Diagnosis and Monitoring

Mass spectrometry is used in clinical settings to detect antibodies in patient sera, aiding in the diagnosis of autoimmune diseases, infectious diseases, and monitoring therapeutic efficacy.

 

Mass spectrometry-based antibody sequence analysis provides efficient and precise methods for antibody research. With the continuous advancement of mass spectrometry technology, its applications in the biomedical field will become more extensive and in-depth, presenting new opportunities and challenges for antibody engineering, drug development, and clinical diagnostics.