Comprehensive Overview of Antibody Sequencing: From Peptide Identification to Full-Length Assembly

Antibody sequencing is a critical technique for determining the amino acid sequence of antibodies. It is extensively applied in antibody drug development, functional engineering, structural modeling, and intellectual property protection. Centered around high-resolution mass spectrometry, this approach reconstructs complete sequences of antibody light chains (LC) and heavy chains (HC) through enzymatic digestion and data-driven assembly. This article systematically outlines the core workflow, essential technical parameters, and application value of antibody sequencing, aiming to enhance researchers’ understanding of its scientific principles and practical relevance.

Fundamental Principles of Antibody Sequencing

Antibody sequencing employs proteomics techniques to directly decode antibody amino acid sequences at the protein level, without relying on RNA templates. In contrast to nucleic acid sequencing, it is not constrained by the physiological state of the cells, making it particularly advantageous for samples lacking cell lines or with degraded RNA.

The core workflow includes:

• Enzymatic digestion to generate peptide fragments.

• Acquisition of MS/MS spectra via LC-MS/MS.

• De novo sequence reconstruction of peptides.

• Full-length assembly of light and heavy chains.

• Annotation and validation of critical functional domains, such as complementarity-determining regions (CDRs).

Core Workflow and Optimization Strategies

1. Multi-Enzyme Synergistic Digestion

(1) Objective: Enhance peptide redundancy and coverage of variable regions.

(2) Commonly used enzymes: Trypsin, Lys-C, Chymotrypsin, Asp-N.

(3) Advantages: Achieves high sequence coverage and reduces blind spots, especially effective in CDR regions.

2. High-Resolution Mass Spectrometry Acquisition

(1) Platform: LC-MS/MS systems with HCD, ETD, and other fragmentation modes.

(2) Key metrics: Mass accuracy (<1 ppm), resolution, dynamic range.

(3) Outcomes: Enables detection of low-abundance peptides and post-translational modifications, ensuring sequence fidelity.

3. Integrated De Novo Sequencing and Database Matching

(1) De novo analysis: Suitable for novel antibody sequences, reconstructs peptides from fragment ion spectra.

(2) Database matching: Increases confidence in identifying framework regions.

(3) Combined advantage: Balances novel sequence discovery with controlled error rates.

4. Full-Length Assembly and Structural Modeling

(1) Assembly criteria: Overlapping peptides, enzymatic cleavage site logic, consistency validation.

(2) Assisted modeling: Utilizes IgG structural templates to delineate framework and CDR boundaries.

(3) Cross-validation: May incorporate transcriptomic or external expression data to enhance reliability.

Quality Control Standards

To ensure scientific validity and practical applicability of antibody sequencing results, the following quality metrics are emphasized:

• Sequence coverage: >95% for full-length chains; complete CDR coverage.

• Assembly consistency: Cross-validation using multi-enzyme datasets to resolve ambiguous peptides.

• CDR identification accuracy: Precise localization of CDR1–CDR3, enabling reliable structural modeling.

• Comprehensive report: Includes digestion maps, sequence assembly diagrams, raw MS data, and FASTA format output.

Typical Application Scenarios for Antibody Sequencing

1. Antibody Therapeutics Development

Full-length sequence information is essential for affinity maturation, humanization, and stability optimization.

2. Recombinant Expression of Hybridoma-Derived Antibodies

Enables recombinant production from non-viable or non-expandable hybridoma cells, improving consistency in manufacturing.

3. Biosimilar Design and Development

Sequencing reference antibodies facilitates functional mimicry and structural refinement, accelerating biosimilar innovation.

4. Patent Filing and Intellectual Property Protection

Sequencing data serve as foundational technical documentation for patent drafting, registration, and legal disclosure.

5. Academic Research and Publication

Sequencing of commercial or custom antibodies enhances reproducibility and methodological transparency in published studies.

Antibody sequencing serves as a crucial bridge between antibody discovery and downstream engineering. Its accuracy significantly impacts both research efficiency and the success of translational and industrial applications. By refining digestion protocols, advancing MS instrumentation, and optimizing assembly algorithms, researchers can achieve complete and reliable antibody sequences, forming a robust foundation for structural characterization, functional enhancement, and therapeutic development. MtoZ Biolabs is committed to delivering high-quality, standardized, and validated antibody sequencing services for the life sciences community. If your project involves missing antibody sequences, undefined functional regions, or patent support needs, feel free to contact us for customized technical solutions and dedicated one-on-one support.

MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.

Related Services

Peptide Sequencing Service