Step-by-Step Guide to Monoclonal Antibody De Novo Sequencing
Why Perform De Novo Sequencing on Monoclonal Antibodies?
In antibody drug development and research, acquiring the full-length amino acid sequence of an antibody is the essential starting point. However, conventional gene sequencing approaches are often not applicable in the following scenarios: 1. The antibody is only available as a purified protein, with no access to B cells or mRNA; 2. The antibody originates from external sources, literature reports, or commercial suppliers, and lacks original sequence data; 3. Clones identified during early-stage R&D or in vivo screening require structural verification through sequence reconstruction; 4. Structural elucidation is necessary for patent applications or consistency evaluations, requiring a directly observable molecular sequence. In such cases, mass spectrometry-based monoclonal antibody De Novo sequencing offers a scientifically robust solution. This approach enables the reconstruction of the complete light chain (LC) and heavy chain (HC) sequences without requiring a reference template, ensuring coverage of the complementarity-determining regions (CDRs) and facilitating subsequent recombinant expression and functional validation.
Overview of the Standard Process for Monoclonal Antibody De Novo Sequencing
1. Antibody Sample Preparation and Quality Assessment
High-purity antibody protein samples are a prerequisite. It is recommended to provide at least 20 µg of antibody solution or a clearly resolved SDS-PAGE band. For samples originating from complex biological matrices such as culture supernatant or serum, purification via Protein A/G affinity chromatography or in-gel PAGE extraction is advised. MtoZ Biolabs offers comprehensive upstream sample preparation services to ensure that samples entering mass spectrometry analysis exhibit high stability, low background noise, and minimal contamination from extraneous proteins.
2. Multi-Enzyme Digestion Strategy Design
Enzymatic digestion critically influences sequence coverage. Relying solely on a single protease such as trypsin may result in inadequate cleavage within hydrophobic or CDR regions, leading to gaps in sequence coverage. To address this, the protocol employs parallel digestion with 3–5 proteases of distinct specificities (e.g., trypsin, chymotrypsin, AspN, LysC, GluC). This combinatorial approach generates a diverse and highly overlapping set of peptides, enhancing coverage and providing a robust foundation for accurate sequence reconstruction.
3. LC-MS/MS High-Resolution Mass Spectrometry Acquisition
Peptides derived from each digestion system are individually subjected to nano-liquid chromatography (NanoLC) separation, followed by tandem mass spectrometry using high-resolution platforms such as Orbitrap Fusion Lumos or timsTOF Pro. Fragmentation modes including HCD, CID, and ETD are employed to acquire detailed MS/MS spectra, generating b and y ion series that serve as critical input data for De Novo sequencing algorithms.
4. De Novo Algorithm Analysis and Sequence Assembly
The acquired MS/MS spectra are processed using widely adopted De Novo sequencing software tools (e.g., PEAKS Studio, Novor, pNovo) to predict peptide sequences. Full-length sequences of the antibody’s light and heavy chains are then assembled based on peptide overlaps, structural template alignment, and manual curation. Functional regions such as the CDRs, framework regions (FRs), and hinge domains are specifically annotated. Ambiguous residues, including isobaric amino acids like isoleucine/leucine (I/L), are further verified through manual inspection.
5. Post-Translational Modification (PTM) Identification and Annotation (Optional)
Monoclonal antibody proteins frequently undergo post-translational modifications (PTMs) such as glycosylation, oxidation, and deamidation. These modifications are critical for informing the selection of expression systems and assessing functional activity. If needed, MtoZ Biolabs can conduct PTM analysis concurrently with sequencing, delivering detailed information on modification sites, modification types, and associated confidence scores.
6. Sequence Validation and Expression Template Construction (Optional)
Following de novo sequence determination, MtoZ Biolabs offers optional services for clients requiring recombinant expression, including:
(1) Reverse translation of the protein sequence into cDNA
(2) Construction and synthesis of expression vectors
(3) Transient transfection and expression in CHO or HEK293 cells
(4) Functional validation via ELISA, Western Blot, and other bioassays
This integrated workflow enables a seamless transition from sequence data to functional antibody reconstruction, ensuring that the sequencing results are experimentally verifiable, recombinantly expressible, and suitable for downstream application.
Advantages of MtoZ Biolabs in Monoclonal Antibody De Novo Sequencing
MtoZ Biolabs has established a rigorous, high-coverage, and high-fidelity system for standardized monoclonal antibody De Novo sequencing. Our key technical strengths include:
1. Optimization of a multi-enzyme digestion system, achieving >95% sequence coverage and >98% full-region CDR identification accuracy
2. Parallel mass spectrometry analysis leveraging Orbitrap and timsTOF platforms to enhance data throughput and precision
3. Proprietary algorithm integrated with manual validation to ensure accurate sequence assembly and prevent light/heavy chain mismatches
4. Comprehensive PTM mapping integrated with sequencing to provide not only primary structure information but also insights into modifications and structural-functional relationships
5. Optional expression verification services enabling a closed-loop workflow from sequencing to function, ensuring reliable and reproducible results
We have successfully supported clients including biopharmaceutical companies, academic institutions, and hospital research groups in antibody reconstruction, restoration of legacy antibodies, affinity screening, and functional validation. These services have accelerated antibody engineering timelines and helped address challenges related to intellectual property and product redevelopment. Structural analysis of monoclonal antibodies should go beyond sequence identification, extending toward functional restoration, rational design, and translational application. As the only methodology capable of directly reconstructing full antibody structures from protein samples, De Novo sequencing serves as a vital bridge between experimental material and expression systems.
MtoZ Biolabs remains committed to delivering dependable, high-quality monoclonal antibody De Novo sequencing services through cutting-edge mass spectrometry platforms, optimized protocols, and expert data interpretation—empowering more efficient, transparent, and controllable antibody development. If you have antibody samples ready for analysis, we welcome you to submit your project details for a customized technical evaluation and solution.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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