Application and Advantages of LC-MS/MS in Antibody Peptide Sequencing

Accurate analysis of antibody primary structure is of great significance in drug development, functional research, and quality control.Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) technologyWith its high sensitivity, wide dynamic range, and ability to recognize post-translational modifications, it has become a key tool for antibody peptide sequencing, providing a high-throughput and reproducible solution for antibody structure analysis.

 

I. Overview of the LC-MS/MS technical process in antibody peptide sequencing

Antibody sequencing involves enzymatic digestion, separation, fragmentation, and data analysis of proteins using mass spectrometry to obtain amino acid sequence information. The main steps include:

• Enzymatic digestion: Cutting the antibody into small peptides suitable for analysis to enhance mass spectrometry coverage;

• Chromatographic separation: Using liquid chromatography to gradient separate peptide mixtures, reducing signal interference;

• Tandem mass spectrometry analysis: Obtaining peptide fragment ion information in MS/MS mode for sequence deduction;

• Data interpretation: Combining database comparison and de novo sequencing methods to complete sequence reconstruction.

 

The entire process does not require a known sequence background, making it suitable for antibody samples from a wide range of sources and structural types.

 

II. Technical advantages of LC-MS/MS in antibody sequencing

1. High sequence coverage

LC-MS/MS can achieve high coverage measurement of both heavy and light chains of antibodies, especially with strong analytical capability for the Complementarity Determining Regions (CDR), helping to reveal key structural differences.

 

2. Strong ability to identify post-translational modifications

Mass spectrometry technology can accurately detect post-translational modifications such as oxidation, deamidation, and glycosylation, enhancing the understanding of antibody functional status and stability.

 

3. Supports de novo sequencing of unknown antibodies

It enables de novo assembly without reference sequences, suitable for antibody samples with unpublicized or unknown sources, broadening the scope of antibody structural research.

 

4. Suitable for consistency assessment and variation detection

By comparing mass spectra, structural differences, modification shifts, or expression system-induced changes between antibody batches can be detected, enhancing process controllability.

 

5. Flexible adaptation to multiple types of antibody structures

LC-MS/MS is applicable to all forms of antibodies, including full-length antibodies, antibody fragments, and complex engineered antibodies, demonstrating good versatility.

 

III. Application scenarios of LC-MS/MS in antibody development

LC-MS/MS is widely used in various stages of antibody drug development, covering all key aspects from screening to quality management:

• Sequence confirmation: Used to verify whether the expressed antibody matches the expected sequence, preventing mutation introduction;

• Structural comparison: Comparing antibody structure consistency across different samples for stability assessment;

• Variation and modification monitoring: Identifying minor amino acid substitutions and PTMs to ensure function is unaffected;

• De novo sequencing applications: Performing structural assembly for antibody molecules lacking public information, suitable for basic research and differentiated development;

• Quality control support: Providing molecular-level structural evidence for process scale-up and drug registration.

The high resolution and reproducibility of LC-MS/MS make it an important technical path for antibody structure analysis.

 

As a core technical means for antibody peptide sequencing, LC-MS/MS has played a key role in various stages of antibody research and development. Its comprehensive advantages in sequence analysis, modification recognition, and consistency evaluation make it an important platform for current antibody structural characterization. In the context of increasingly complex antibody drugs and stricter structural requirements, choosing a service team with professional technical capabilities and high-quality delivery standards is crucial for ensuring project progress and data compliance. BioAccord Biotech Co., Ltd. specializes in high-resolution mass spectrometry analysis and looks forward to providing reliable data support and technical assurance for your antibody development projects.

 

BioAccord Biotech Co., Ltd. - Characterization of Biologics, a high-quality service provider for multi-omics biomass spectrometry detection

 

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