Antibody Sequencing Service | De Novo & LC-MS/MS

Antibody Sequencing Service

Antibody sequencing service determines antibody heavy chain, light chain, variable region, and CDR sequence information using protein-level or genetic workflows. It helps researchers characterize unknown, commercial, monoclonal, engineered, or hybridoma-derived antibodies when sequence confirmation is needed for discovery, validation, biosimilar research, or antibody engineering.

De novo, LC-MS/MS, and genetic workflow-supported antibody sequencing solutions for monoclonal antibody characterization, CDR identification, and heavy/light chain sequence analysis.

De Novo Sequencing LC-MS/MS Analysis CDR Identification PCR / NGS Support

Service Scope

Sequencing workflows for unknown, commercial, monoclonal, and engineered antibodies

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MS-Based De Novo Sequencing

Recover antibody sequence information directly from protein samples.

Heavy/Light Chain Analysis

Support VH, VL, framework region, and CDR interpretation.

CDR Annotation

Identify antigen-binding regions for engineering and validation projects.

Genetic Workflows

Use PCR, Sanger, or NGS-supported approaches when genetic samples are available.

Unknown

Support for antibodies without available sequence or hybridoma information.

Commercial

Protein-level sequencing for commercial antibody validation projects.

Engineered

Sequence information for humanization, CDR analysis, and engineering workflows.

Method Selection

Services

Workflow

Platform

Advantages

Samples

Deliverables

Case Study

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What Problems Does Antibody Sequencing Solve?

Creative Proteomics provides comprehensive antibody sequencing services for purified antibodies, commercial antibodies, monoclonal antibodies, hybridoma-derived antibodies, recombinant antibody fragments, and antibody engineering projects.

Our service primarily uses LC-MS/MS-based de novo antibody sequencing, supported when appropriate by PCR-based sequencing, Sanger sequencing, NGS-supported variable region sequencing, Edman degradation for selected N-terminal confirmation, and peptide mapping strategies. This helps support antibody discovery, validation, biosimilar research, sequence confirmation, and CDR analysis.

Antibody sequencing helps researchers answer practical questions that directly affect antibody development, validation, and downstream engineering. Instead of only asking what the antibody is, researchers often need to know whether the antibody can be reproduced, engineered, compared, or validated at the sequence level.

• Unknown antibody sequence recovery when no sequence record is available
• Lost hybridoma or unavailable cell line projects where genetic material cannot be accessed
• Commercial antibody validation to confirm sequence identity or support reproducibility studies
• Monoclonal antibody characterization for research, diagnostic, or therapeutic development
• Biosimilar and biobetter research requiring sequence-level comparison
• Antibody humanization and engineering based on VH, VL, and CDR information
• CDR identification to support antigen-binding region analysis
• Protein-level confirmation when DNA-based sequencing alone is insufficient

Antibody sequencing is especially valuable when the antibody protein is available but the original antibody-producing cells, plasmids, or genetic records are missing.

Antibody sequencing workflow concept showing antibody protein-level and genetic sequencing routes.

Antibody Sequencing Methods Overview

Creative Proteomics emphasizes mass spectrometry-based antibody sequencing because it can work directly from antibody protein. Other methods, including PCR-based sequencing, Sanger sequencing, NGS-supported sequencing, Edman degradation, and peptide mapping, can also be useful depending on sample type and project objective.

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Unknown Antibody Recovery

Antibody sequencing can recover antibody sequence information when hybridoma cells or DNA are unavailable.

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MS-Based Main Workflow

LC-MS/MS-based de novo antibody sequencing is suitable for purified or commercial antibodies.

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Genetic Workflow Support

PCR, Sanger, or NGS-supported sequencing may be useful when viable hybridoma cells, B cells, or RNA are available.

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CDR Interpretation

CDR sequencing helps identify antigen-binding regions that determine antibody specificity.

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Evidence-Based Reporting

A complete antibody sequencing report should include heavy/light chain sequences, peptide evidence, CDR annotation, coverage information, and confidence assessment.

Method	Starting Material	Best Use Case	Key Limitation
LC-MS/MS-based de novo sequencing	Purified antibody protein	Unknown, commercial, or lost-hybridoma antibody projects	Requires complex MS data interpretation
PCR-based antibody sequencing	Hybridoma cells, B cells, RNA, or other genetic material	Hybridoma-derived antibodies or antibody-producing cells	Requires viable or usable genetic material
Sanger sequencing	PCR amplicons or cloned antibody variable regions	Confirming VH/VL coding sequences from defined clones	Not suitable for complex mixtures without prior amplification or cloning
NGS-supported sequencing	B-cell repertoires or amplified antibody libraries	High-throughput repertoire or discovery projects	Requires genetic material and bioinformatics filtering
Edman degradation	Purified protein or peptide with accessible N-terminus	Selected N-terminal confirmation	Limited read length and blocked N-termini can prevent sequencing
Peptide mapping	Purified antibody protein with known or expected sequence	Sequence confirmation, comparability, and coverage analysis	Usually supports confirmation rather than complete unknown sequence discovery alone

Practical conclusion: LC-MS/MS-based de novo antibody sequencing is the preferred method when only the antibody protein is available. PCR-based, Sanger, or NGS-supported sequencing may be preferred when high-quality genetic material is available.

Which Antibody Sequencing Method Should You Choose?

Different antibody projects require different sequencing strategies. The table below helps researchers select a suitable workflow before submitting samples.

Project Scenario	Recommended Method	Why This Method Fits
Unknown antibody with no DNA or hybridoma	LC-MS/MS-based de novo sequencing	Directly reconstructs sequence from purified antibody protein
Commercial antibody sequence validation	LC-MS/MS-based antibody sequencing	Works from the antibody product itself
Lost hybridoma but purified antibody available	LC-MS/MS-based de novo sequencing	Does not require viable antibody-producing cells
Hybridoma cells available	PCR-based sequencing followed by Sanger or NGS-supported confirmation	Retrieves VH and VL coding sequences from genetic material
B-cell repertoire or antibody library project	NGS-supported antibody sequencing	Supports high-throughput sequence discovery and repertoire analysis
CDR identification for antibody engineering	LC-MS/MS, PCR-based, or NGS-supported sequencing	Supports variable region and CDR annotation
Modified or conjugated antibody analysis	LC-MS/MS-based characterization	Can provide protein-level information on modifications when included in the scope
Biosimilar or antibody comparability study	LC-MS/MS sequencing plus peptide mapping	Supports sequence confirmation and peptide-level evidence
N-terminal sequence confirmation	Edman degradation or LC-MS/MS-based peptide confirmation	Supports selected terminal confirmation when the project scope is limited

Comprehensive Antibody Sequencing Services

Creative Proteomics provides integrated antibody sequencing solutions for different project types, sample formats, and research objectives.

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De Novo Antibody Sequencing

Our de novo antibody sequencing service reconstructs antibody amino acid sequences directly from protein samples without relying on prior DNA or complete database information. This workflow is suitable for unknown monoclonal antibodies, commercial antibodies, and antibodies whose original hybridoma or coding sequence is unavailable.

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LC-MS/MS-Based Antibody Sequencing

LC-MS/MS-based antibody sequencing uses enzymatic digestion, tandem mass spectrometry, and bioinformatics-based peptide assembly to infer antibody sequences. This approach is especially useful for protein-level sequence confirmation and CDR-supporting peptide evidence.

LC-MS/MS De Novo

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Heavy and Light Chain Sequencing

We support heavy chain and light chain sequence analysis, including VH, VL, framework regions, and CDR annotation. When needed, chain separation and chain-specific peptide evidence can be used to improve sequence interpretation.

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CDR Sequencing and Annotation

CDRs are the primary antigen-binding regions of antibodies. Our antibody sequencing workflow can support CDR identification, annotation, and sequence-level interpretation for antibody engineering, humanization, binding studies, and validation projects.

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PCR, Sanger, and NGS-Supported Antibody Sequencing

When hybridoma cells, B cells, RNA, PCR amplicons, or antibody libraries are available, genetic workflows can support VH/VL coding sequence analysis, clone confirmation, or repertoire-level discovery.

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Terminal and Peptide-Level Confirmation

For selected projects, Edman degradation, peptide mapping, and targeted LC-MS/MS confirmation can support terminal sequence checks, coverage review, or sequence comparability analysis.

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How Antibody Sequencing Works

Antibody sequencing projects are customized according to sample type, antibody format, and project goal. A typical LC-MS/MS-based de novo antibody sequencing workflow includes the following steps.

Project Evaluation

The antibody type, sample format, purity, quantity, species, antibody subclass, and sequencing objective are reviewed to determine the most suitable strategy.

Sample Preparation

Purified antibodies may be reduced, alkylated, cleaned up, deglycosylated, enriched, or separated into heavy and light chains depending on project needs.

Multi-Enzyme Digestion

Multiple proteases may be used to generate overlapping peptides. This improves sequence coverage and helps resolve difficult regions.

LC-MS/MS Analysis

Peptides are analyzed using tandem mass spectrometry to generate precursor and fragment ion data.

Report Delivery

A final report is provided with sequence results, coverage information, peptide evidence, CDR annotation, confidence notes, and expert interpretation.

De Novo Peptide Interpretation

MS/MS spectra are interpreted to identify peptide sequences without requiring a complete reference sequence.

Sequence Assembly

Overlapping peptide evidence is used to reconstruct heavy chain, light chain, variable region, framework region, and CDR sequences.

Sequence Validation

Key regions, ambiguous residues, high-value peptides, CDRs, and possible modifications are reviewed to improve confidence.

Technology Platform for Antibody Sequencing

Laboratory mass spectrometry platform for antibody sequencing and peptide analysis.

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LC-MS/MS-Based Protein Sequencing Platform

Tandem mass spectrometry supports peptide identification, de novo sequencing, peptide evidence review, coverage analysis, and sequence validation for antibody protein samples.

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Multiple Protease Digestion Strategy

Antibody sequence reconstruction often requires overlapping peptides from different digestion strategies. Proteases such as trypsin, chymotrypsin, Glu-C, Asp-N, Lys-C, or other enzymes may be selected depending on antibody sequence complexity and project goals.

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Complementary Fragmentation Strategies

Different fragmentation methods may provide complementary peptide information. When needed, fragmentation strategy optimization can support improved sequence confidence, especially in variable regions and CDR-containing peptides.

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Bioinformatics-Based Sequence Assembly

De novo antibody sequencing requires careful interpretation of peptide-level data. Bioinformatics analysis supports peptide assembly, sequence alignment, coverage evaluation, CDR annotation, and ambiguity reporting.

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CDR and Variable Region Annotation

For antibody engineering and functional analysis, VH, VL, framework regions, and CDRs can be annotated in the final report to help researchers interpret sequence-function relationships.

Technical Challenges and Our Solutions

Antibody sequencing is technically demanding because antibodies contain variable regions, highly similar sequence motifs, post-translational modifications, and chain-specific complexity. The table below summarizes common challenges and how Creative Proteomics addresses them.

Technical Challenge	Why It Matters	Our Solution
Heavy/light chain complexity	Peptides from different chains must be assigned correctly	Chain separation, peptide mapping, and chain-specific evidence review
CDR sequence complexity	CDRs are highly variable and functionally important	Complementary digestion and careful CDR-supporting peptide validation
Incomplete sequence coverage	Some regions may produce weak or missing peptides	Use complementary enzymes and optimized LC-MS/MS acquisition
Leucine/isoleucine ambiguity	Leu and Ile have identical mass and can be difficult to distinguish by standard MS/MS	Report ambiguity transparently and use orthogonal evidence when available
PTMs or conjugation	Modifications may affect digestion, ionization, or peptide interpretation	Apply PTM-aware analysis and customized sample preparation
Low sample amount	Insufficient material may reduce peptide coverage	Optimize cleanup, digestion, and MS sensitivity
Buffer incompatibility	Detergents, salts, glycerol, or additives can interfere with MS	Recommend buffer exchange or cleanup before analysis
Commercial antibody complexity	Formulation excipients may interfere with analysis	Review datasheets and perform feasibility-based preparation

Factors That Affect Antibody Sequencing Success

Antibody sequencing success depends on both sample quality and project design. For this reason, Creative Proteomics evaluates each project before recommending a final workflow.

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Sample purity

Higher purity generally supports better peptide detection and cleaner sequence assembly.

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Sample amount

Sufficient antibody material improves digestion, repeated analysis, and validation of difficult regions.

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Buffer composition

Detergents, salts, glycerol, stabilizers, preservatives, and carrier proteins may interfere with LC-MS/MS analysis.

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Antibody format

Full-length IgG, Fab, scFv, nanobody, conjugated antibody, and bispecific antibody formats may require different strategies.

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Heavy/light chain separation

Chain separation can improve confidence when heavy and light chain peptide assignment is challenging.

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Sequence complexity

Highly homologous antibodies, unusual CDRs, or modified regions may require additional interpretation.

Advantages of Creative Proteomics Antibody Sequencing Service

Creative Proteomics provides antibody sequencing solutions designed for research teams that need reliable, interpretable, and project-specific results.

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Direct Sequencing Without Prior DNA

LC-MS/MS-based de novo antibody sequencing can be performed directly from purified antibody protein, making it suitable for unknown antibodies, commercial antibodies, and lost hybridoma projects.

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Flexible Sequencing Strategies

We support LC-MS/MS-based workflows as the primary route, with PCR-based, Sanger, NGS-supported, Edman, and peptide mapping strategies available when they fit the sample and objective.

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CDR-Focused Interpretation

Because CDRs are critical for antigen binding, our workflow emphasizes variable region and CDR annotation where sufficient sequence evidence is available.

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Heavy and Light Chain Analysis

Our antibody sequencing service supports heavy chain and light chain analysis, including chain-specific sequence interpretation and reporting.

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Customized Project Design

Each project is evaluated based on antibody type, available material, sample purity, and desired deliverables.

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Expert Bioinformatics Review

Antibody sequencing data require expert interpretation. Our team reviews peptide evidence, sequence coverage, ambiguity, and CDR-related information to deliver a practical final report.

Sample Requirements for Antibody Sequencing

Sample requirements vary depending on antibody type, workflow, and project objective. The table below provides general guidance. Final requirements should be confirmed during project evaluation.

Sample Type	Recommended Use	Notes
Purified monoclonal antibody	LC-MS/MS-based de novo sequencing	Preferred sample type for protein-level antibody sequencing
Commercial antibody	Sequence validation or unknown sequence recovery	Provide datasheet, formulation, host species, clone name, and concentration if available
Hybridoma cells	PCR-based antibody sequencing	Suitable when viable cells or high-quality genetic material are available
B cells or RNA	PCR-based or NGS-supported antibody sequencing	Requires appropriate sample preservation and RNA quality
PCR amplicon or plasmid	Sanger sequencing or NGS-supported confirmation	Useful for confirming defined VH/VL coding regions
Antibody in serum or plasma	Targeted antibody enrichment before sequencing	Feasibility evaluation is required due to high sample complexity
Conjugated antibody	Customized LC-MS/MS workflow	Provide conjugation chemistry, linker information, and labeling ratio if known
Fab, scFv, nanobody, or recombinant fragment	Protein or genetic sequencing depending on sample	Workflow depends on format and available material

Applications of Antibody Sequencing

Antibody sequencing supports a wide range of antibody research, development, and validation applications.

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Monoclonal Antibody Characterization

Sequence information helps characterize monoclonal antibodies at the protein or genetic level and supports reproducibility.

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Commercial Antibody Validation

Commercial antibodies can be sequenced to support identity confirmation, reproducibility assessment, or research tool validation.

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Lost Hybridoma Sequence Recovery

When antibody-producing cells are unavailable, LC-MS/MS-based de novo sequencing can help recover sequence information from the antibody protein.

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Antibody Humanization

VH, VL, and CDR information provides a foundation for antibody humanization and engineering workflows.

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Biosimilar and Biobetter Research

Sequence-level information supports comparability studies and antibody candidate evaluation.

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Antibody Engineering

Sequence information supports affinity maturation, recombinant antibody design, format conversion, and expression construct development.

Example antibody sequencing report deliverables including sequence coverage and CDR annotation.

Deliverables of Antibody Sequencing Analysis

Creative Proteomics provides clear, project-specific deliverables to support downstream research and decision-making. Typical deliverables may include:

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Sequence Information

Full or partial antibody amino acid sequence, heavy chain sequence information, and light chain sequence information.

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VH, VL, and CDR Annotation

VH and VL region annotation plus CDR identification and annotation.

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Peptide Evidence

Peptide coverage map, peptide evidence table, and MS/MS spectral evidence summary.

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Confidence Assessment

Sequence confidence assessment and notes on ambiguous residues such as Leu/Ile where applicable.

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Modification Information

PTM or modification information when included in the project scope.

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Expert Interpretation

Method summary, data interpretation, and expert recommendations for follow-up validation if needed.

Case Example

Reverse-Engineering the Anti-MUC1 Antibody 139H2 by Mass Spectrometry-Based De Novo Sequencing

Sample

Hybridoma-derived 139H2 IgG

Prep

Protein G purification and multi-protease digestion

Platform

LC-MS/MS with sHCD and EThcD

Output

Full-length heavy and light chain reconstruction

Project Overview

The anti-MUC1 monoclonal antibody 139H2 is a widely used research antibody against the MUC1 variable number tandem repeat region. In the reported study, 139H2 IgG was purified from hybridoma culture supernatant, sequenced directly by bottom-up mass spectrometry, and reconstructed as a functional recombinant monoclonal antibody.

The work demonstrates how direct protein-level sequencing can help recover valuable monoclonal antibody reagents, support recombinant re-expression, and improve long-term reproducibility when an antibody sequence is not readily available.

Background

MUC1 is a transmembrane mucin with a variable number tandem repeat region that is heavily O-glycosylated.
Monoclonal antibodies against MUC1 are useful research tools for studying MUC1 biology and MUC1-expressing cancers.
The 139H2 antibody recognizes the MUC1 VNTR region and was selected for sequence recovery and recombinant reconstruction.
Direct MS-based sequencing was used to obtain heavy and light chain sequence information from the antibody protein.

Analytical Challenge

The sequence of a valuable hybridoma-derived antibody needed to be recovered at the protein level.
Antibody heavy and light chains required confident reconstruction from LC-MS/MS peptide evidence.
Overlapping peptides from multiple proteases were needed to support sequence assembly.
The reconstructed antibody needed functional validation after recombinant expression.

Workflow

Sample Type

139H2 IgG purified from hybridoma culture supernatant

Sample Preparation

Protein G affinity purification followed by in-solution digestion

Protease Strategy

Parallel digestion with trypsin, chymotrypsin, alpha-lytic protease, and thermolysin

Mass Spectrometry Analysis

LC-MS/MS using stepped high-energy collision dissociation and electron-transfer high-energy collision dissociation

Data Analysis

Peptide sequences predicted from MS/MS spectra using PEAKS and assembled with Stitch

Validation

Recombinant 139H2 expression and structural analysis of the Fab fragment bound to the MUC1 epitope

Key Insight

Mass spectrometry-based de novo sequencing enabled reconstruction of the heavy and light chain sequences of a hybridoma-derived monoclonal antibody. The reconstructed sequence supported recombinant antibody production and downstream structural characterization.

Service Value

Supports sequence recovery from antibody protein when sequence records are unavailable
Uses multiple proteases to improve overlapping peptide evidence
Provides heavy and light chain sequence reconstruction for recombinant antibody development
Helps improve antibody reagent reproducibility and long-term usability

Key Findings

Sequence reconstruction, recombinant expression, and functional validation

139H2

Antibody

139H2

Target

MUC1

Workflow

LC-MS/MS

The study reported direct mass spectrometry-based sequencing of anti-MUC1 hybridoma-derived 139H2 IgG, full-length heavy and light chain reconstruction, recombinant antibody expression, and structural characterization of the 139H2 Fab fragment in complex with the MUC1 epitope.

Reference

Reverse-engineering the anti-MUC1 antibody 139H2 by mass spectrometry-based de novo sequencing

PubMed record: Reverse-engineering the anti-MUC1 antibody 139H2 by mass spectrometry-based de novo sequencing

Frequently Asked Questions

Can you sequence an antibody without hybridoma cells?expand_more

Yes. If purified antibody protein is available, LC-MS/MS-based de novo antibody sequencing can be used without hybridoma cells, DNA, or RNA. This is suitable for unknown antibodies, commercial antibodies, and lost hybridoma projects.

Can you sequence a commercial antibody?expand_more

Yes. Commercial antibodies can often be analyzed by LC-MS/MS-based antibody sequencing if sufficient material and acceptable sample quality are available. Providing the datasheet, clone name, host species, concentration, formulation, and antibody type helps with project evaluation.

What is de novo antibody sequencing?expand_more

De novo antibody sequencing reconstructs antibody amino acid sequences without relying on a complete reference DNA or protein sequence. In LC-MS/MS-based workflows, antibody proteins are digested into peptides, analyzed by tandem mass spectrometry, and assembled into heavy and light chain sequences.

What is the difference between LC-MS/MS-based and PCR-based antibody sequencing?expand_more

LC-MS/MS-based antibody sequencing starts from antibody protein and does not require genetic material. PCR-based antibody sequencing starts from hybridoma cells, B cells, RNA, or other genetic material. LC-MS/MS is preferred when only purified antibody is available, while PCR-based sequencing is preferred when viable antibody-producing cells are available.

How do Sanger sequencing and NGS fit into antibody sequencing?expand_more

Sanger sequencing can confirm amplified or cloned VH/VL sequences from defined antibody sources. NGS-supported antibody sequencing can support B-cell repertoire, antibody library, and discovery projects when genetic material is available.

Can you identify CDR regions?expand_more

Yes. CDR identification and annotation can be included when sufficient sequence information is obtained from heavy and light chain variable regions. CDR analysis is especially useful for antibody engineering, humanization, and antigen-binding studies.

Can you sequence both heavy and light chains?expand_more

Yes. Heavy and light chain sequence analysis can be performed using LC-MS/MS-based or genetic workflows. Chain-specific interpretation may require chain separation, overlapping peptide evidence, or genetic material depending on the project.

What sample type gives the best result?expand_more

Purified monoclonal antibody is generally the preferred sample type for LC-MS/MS-based de novo antibody sequencing. For PCR-based, Sanger, or NGS-supported sequencing, high-quality hybridoma cells, B cells, RNA, amplicons, or library material may be preferred.

Can antibody sequencing identify PTMs or modifications?expand_more

LC-MS/MS-based workflows can support protein-level modification analysis when included in the project scope. However, modification detection depends on sample quality, modification type, peptide coverage, and analytical strategy.

Can you distinguish leucine and isoleucine?expand_more

Leucine and isoleucine have the same mass, so they can be difficult to distinguish by standard MS/MS. When residue-level differentiation is critical, additional strategies or orthogonal evidence may be required. Ambiguities should be clearly reported in the final results.

What causes incomplete antibody sequence coverage?expand_more

Incomplete coverage can result from low sample amount, poor purity, difficult peptide behavior, incompatible buffer components, modifications, glycosylation, conjugation, or regions that do not generate suitable peptides under a given digestion condition.

What will be included in the final report?expand_more

The final report may include heavy and light chain sequences, VH/VL annotation, CDR annotation, peptide evidence, sequence coverage maps, confidence assessment, ambiguous residue notes, PTM information if applicable, and expert interpretation.

Creative Proteomics provides antibody sequencing services for research use only. These services are not intended for clinical diagnosis, treatment, or direct therapeutic decision-making.