De Novo Antibody Sequencing Service Using High-Resolution LC-MS/MS

Unlocking Antibody Insights with De Novo Sequencing

In today's fast-moving landscape of therapeutic antibody discovery and protein engineering, researchers are increasingly faced with a critical challenge: how to recover or confirm the precise amino acid sequence of an antibody when no genetic template is available. Whether due to hybridoma loss, proprietary constraints, or incomplete sequence records, the need to reconstruct antibody sequences from scratch—de novo—has become a pivotal step in many biopharma and CRO workflows.

De novo antibody sequencing uses advanced mass spectrometry (MS)-based techniques to directly read peptide fragments from purified antibodies. This bypasses the need for DNA or mRNA templates, enabling scientists to recover heavy and light chain variable regions, especially complementarity-determining regions (CDRs), with high accuracy. As patent expirations, biosimilar development, and antibody repurposing expand across the industry, this technology is increasingly vital for:

• Generating recombinant antibody sequences from legacy or unknown sources
• Supporting antibody humanization and optimization strategies
• Ensuring functional integrity during antibody reformatting and QC
• Enabling regulatory documentation or IP filings for novel biologics

From global pharma teams working on next-generation biologics to academic labs salvaging precious hybridoma lines, de novo sequencing empowers scientists to regain control over their antibody assets—accurately, reproducibly, and without genetic constraints.

What Is De Novo Antibody Sequencing?

De novo antibody sequencing refers to the process of determining an antibody's amino acid sequence directly from the protein itself—without relying on existing genetic or sequence information. Unlike template-based sequencing, which aligns peptide fragments against known reference databases, de novo sequencing builds the sequence from scratch, using only the experimental data obtained from tandem mass spectrometry (MS/MS).

In protein research, "de novo" literally means "from the beginning." This method is particularly critical when:

• The original hybridoma cell line is lost, degraded, or contaminated
• No cDNA or mRNA is available for traditional sequencing
• The antibody originates from proprietary, non-disclosed sources
• Accurate sequence confirmation is required for biosimilar development or intellectual property filing

For antibodies, de novo sequencing must resolve not only the overall chain composition, but also the hypervariable CDR regions, which define antigen-binding specificity. This requires high-coverage MS data, careful peptide fragmentation (often via multiple digestion enzymes), and sophisticated software algorithms that can reconstruct overlapping peptide maps to assemble the full heavy (VH) and light (VL) chain sequences.

The end result is a template-independent, research-grade antibody sequence that enables recombinant expression, epitope mapping, functional studies, and more. It's a powerful rescue and validation tool for anyone working with unknown or legacy antibodies.

Our De Novo Antibody Sequencing Service

At Creative Proteomics, we offer flexible and accurate de novo antibody sequencing with minimal sample requirements. In most cases, just a few hundred micrograms of purified antibody—≥ 95% purity—is sufficient for full heavy and light chain sequence determination.

Our platform integrates multi-enzyme digestion strategies with high-resolution LC-MS/MS and proprietary databases to confidently differentiate closely related amino acids such as leucine and isoleucine. These distinctions are made based on enzyme-specific cleavage patterns and established frequency data from our in-house reference sets.

Beyond primary sequence coverage, we also provide detailed characterization of post-translational modifications (PTMs)—including glycosylation, oxidation, and other user-defined modifications. These insights are critical for antibody functional studies, expression stability assessments, or biosimilar comparison projects.

How Does De Novo Antibody Sequencing Work?

1. Sample Preparation

The process begins with a purified antibody sample, typically in the form of intact IgG or Fab fragments. To enhance sequence coverage, the antibody is digested using multiple complementary proteases (e.g., trypsin, chymotrypsin, Lys-C) to generate overlapping peptides that span the full sequence.

2. Peptide Fragmentation via Tandem MS

Each resulting peptide is subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Inside the instrument, peptides are ionized and fragmented using methods such as:

• Collision-Induced Dissociation (CID)
• Higher-energy Collisional Dissociation (HCD)
• Electron Transfer Dissociation (ETD)

These fragmentation strategies break peptides at different backbone locations, creating b- and y-ion series that reveal the amino acid sequence.

3. De Novo Peptide Assembly

Using sophisticated algorithms, de novo sequencing software analyzes MS/MS spectra to predict short peptide sequences (known as "tags") directly from the fragmentation data. These tags are then aligned and stitched together based on overlapping regions, enzyme specificity, and mass constraints.

4. Full-Length Sequence Reconstruction and Validation

The assembled peptide maps are used to reconstruct the complete heavy and light chain sequences, including variable (V), diversity (D), joining (J), and constant (C) regions where applicable. Manual curation is often employed to confirm critical regions like CDRs and to resolve ambiguous residues such as isoleucine/leucine.

When needed, additional fragmentation methods or orthogonal validation techniques may be used to ensure high sequence confidence.

Applications in Biopharma and CROs

✅ Recombinant Antibody Production from Lost or Legacy Sources

One of the most common use cases is recreating antibodies from hybridomas that are no longer viable or have lost productivity due to genetic drift or contamination. De novo sequencing enables the accurate reconstruction of heavy and light chain sequences, which can then be cloned and expressed recombinantly—ensuring consistent performance across batches.

✅ Biosimilar Development and Intellectual Property Strategy

Pharmaceutical companies developing biosimilars often require precise characterization of originator antibodies—especially when sequence data is not publicly disclosed. De novo sequencing provides high-confidence results that support:

• Competitive product profiling
• Patent circumvention or IP documentation
• Regulatory submissions requiring structural comparability

✅ Epitope Mapping and Antibody Humanization

When working with therapeutic candidates derived from animals, researchers often use de novo sequencing to guide CDR grafting and humanization efforts. Knowing the full sequence allows rational design of mutations to reduce immunogenicity while preserving target binding.

✅ Contract Research and QC for Outsourced Projects

CROs offering antibody production or engineering services may need to validate client-delivered antibodies for sequence fidelity, especially when working with legacy reagents or partially annotated sequences. De novo sequencing provides a definitive reference, enabling quality assurance, batch reproducibility, and traceability.

Whether you're recovering a legacy clone or de-risking biosimilar development, de novo sequencing ensures you know exactly what you're working with—amino acid by amino acid.

Service Features: Why Our De Novo Sequencing Delivers

Ultra-High Accuracy

• Distinguishes difficult amino acid pairs (e.g., Leu vs. Ile, Gln vs. Lys)
• Quantifies post-translational modifications and variant forms

Comprehensive Sequence Coverage

• Uses up to 8 proteases to ensure full antibody coverage
• 10× peptide reads for each CDR site to guarantee precision

Compatible with All Antibody Types

• Works with IgG, IgM, and non-standard formats
• Accurate sequencing of labeled antibodies (e.g., FITC, Biotin, GST-tagged)

Reliable for Any Species or Isoform

• Supports antibodies from human, mouse, rabbit, rat, camelid, and more
• Handles different antibody isoforms and subclasses with ease

Rigorous Quality Control

• Manual verification of key residues
• Strict validation from digestion to sequence assembly

Sample Requirements and Submission Guidelines

Sample Formats Accepted

• Purified antibody: IgG, Fab, or scFv
• Supernatant: Hybridoma culture media
• Serum/plasma-derived antibodies
• Labeled antibodies (e.g., FITC, Biotin, GST-tagged)

Minimum Quantity & Purity

Use concentration methods (e.g., Protein A/G) for dilute samples.

Handling, Storage & Shipping

• Immediately freeze samples in liquid nitrogen post-preparation; store at –80 °C.
• Ship on dry ice to maintain low temperatures and avoid freeze–thaw cycles.
• Clearly label each tube with sample ID, concentration, buffer, date.

✅ Tips to Ensure Quality

• Use one species or sample per tube to prevent cross-contamination.
• For antibodies expressed in non-native systems: provide expression host details.
• Ensure supernatant/supplement buffer components are declared (e.g., serum proteins).
• For multi-protease digestion: we handle the process—no on-sample digestion needed beforehand.

Deliverables: What You'll Receive from Our Service

Full-Length Heavy & Light Chain Sequences

• Complete amino acid sequences in FASTA format (VH and VL variable regions plus constant regions)
• Both heavy and light chain alignments, ready for cloning or expression
• Confirmed sequence coverage supporting research-grade applications

Sequence Coverage Maps & Confidence Scores

• Interactive peptide-by-peptide mapping showing depth of coverage (>10× in CDRs and >5× elsewhere)
• Confidence scores for each peptide and residue, including flagged ambiguities
• Data visualization to facilitate in silico engineering and quality validation

Post-Translational Modification & Label Annotations

• Identification of relevant PTMs (e.g., glycosylation, oxidation, deamidation)
• Reporting of any small labels (e.g., FITC, Biotin) or fusion tags (e.g., GST) incorporated in the sample

Raw MS/MS Data (Optional)

• Upon request, deliverables can include all raw LC–MS/MS spectra and result files
• Enables integration into client pipelines, additional analysis, or reprocessing

Recombinant Antibody Expression (Optional Add-On)

• Optionally, we can perform expression and purification of the identified sequence
• Receive recombinant antibody protein—fully validated and ready for downstream use

FAQs: De Novo Antibody Sequencing Clarified

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Case Study: De Novo Sequencing Identifies Functional Antifungal Protein Sin a 1 from Mustard Seeds

Client Institution: Munster Technological University & University College Cork
Publication: Biochemistry and Biophysics Reports, 2022
DOI: 10.1016/j.bbrep.2022.101208

• Background
• Methods
• Key Results
• Conclusion

A research team aimed to isolate and functionally characterize natural antifungal proteins from white mustard (Brassica hirta) seeds. After a series of protein purification steps, they obtained a ~14 kDa protein with strong anti-yeast activity. However, the amino acid sequence of this protein was unknown, and no reference genome or cDNA data were available for direct gene-based identification.

To proceed with sequence validation and isoform comparison, the team required accurate, full-length de novo protein sequencing from the purified sample.

Creative Proteomics' Contribution

To support this discovery, Creative Proteomics performed full-service de novo sequencing of the purified antifungal protein:

• In-gel digestion of SDS-PAGE bands using six complementary proteases (Trypsin, Chymotrypsin, Glu-C, Arg-C, Lys-N, Pepsin)
• High-resolution nano LC-MS/MS analysis of digested peptide fragments
• De novo sequence assembly and analysis using PEAKS Studio v8.5 software
• Final sequence output in FASTA format, suitable for BLASTp homology searches and multiple sequence alignments

This approach provided the client with a complete and confident amino acid sequence, enabling functional annotation and classification.

Protein Extraction: Ammonium sulfate precipitation, thermal denaturation, and cation-exchange chromatography on AKTA system

Purity Check: SDS-PAGE under reducing and non-reducing conditions revealed a 14 kDa protein consisting of two disulfide-linked chains

De Novo Sequencing: Outsourced to Creative Proteomics—full-length sequence obtained (see Figure 3 of publication)

Bioinformatics: BLASTp search identified 96% identity with Sin a 1.0106, a 2S albumin family member

Antimicrobial Testing: MICs determined against yeasts, moulds, and bacteria

Mechanistic Assays: Yeast colony count, membrane permeability (PI staining), nucleotide leakage

Safety & Stability Tests: Caco-2 cytotoxicity, red blood cell hemolysis, resistance to proteases, heat, pH, and salt stress

Key Finding	Details
Sequence Identity	96% match to Allergen Sin a 1.0106 from Sinapis alba
Protein Identity	Confirmed as a Napin family isoform with antifungal properties
Spectrum of Activity	Active against Z. bailii, S. cerevisiae, F. culmorum; no antibacterial effect
Mechanism of Action	Membrane permeabilization and nucleotide leakage confirmed
Biostability	Retains antifungal activity after heat (100°C), pH shifts, and digestion
Safety	Non-toxic to human intestinal cells and red blood cells

Figure: Full-length amino acid sequence of the Sin a 1 protein as identified by Creative Proteomics' de novo LC-MS/MS sequencing. This enabled downstream homology analysis and functional validation.

Thanks to the de novo protein sequencing platform provided by Creative Proteomics, the research team:

• Successfully identified the protein as a functional isoform of Allergen Sin a 1
• Published the report of antifungal activity for this protein in a peer-reviewed journal
• Confirmed that plant-derived storage proteins can serve as promising antifungal agents
• Gained a high-purity, heat-stable, and non-toxic candidate with potential applications in agriculture or food biopreservation

Demo

Publication

Here are some publications in protein sequencing from our clients: