PEAKS AB Polyclonal Antibody
de novo Sequencing Service

Polyclonal antibodies play a critical role in human immunity and their characterisation is important for development of novel therapeutics and disease treatment. They have potential advantages over monoclonal antibody therapeutics, such as higher neutralisation capacity, lower susceptibility of pathogen resistance, and higher avidity. Thus, identifying polyclonal antibodies responsible for antigen neutralisation is essential for improving antibody therapeutics.

Polyclonal antibodies are secreted by different B cell lineages in response to multiple B cell epitopes on a specific antigen. A nearly random recombination of variable (V), diversity (D) and joining (J) gene segments, which occurs during the maturation of B cells in bone marrow, results in a highly diverse repertoire of naturally occurring antibodies and novel amino acid sequences in antigen-binding regions. In contrast to this exceptional diversity, currently available sequence databases for antibodies are extremely limited. Mass spectrometry and de novo protein sequencing have thus become critical for antibody discovery and characterisation.

Unlike sequencing monoclonal antibodies, de novo sequencing of polyclonal antibodies presents a much greater challenge due to the complexity of sequence variants. Traditional bottom-up proteomics approaches used in monoclonal antibody sequencing cannot distinguish between each antibody variant by peptide assembly. To overcome this challenge, the combination of intact, top-down, and bottom-up proteomics data is required. We have developed a complete workflow that integrates enhanced liquid chromatography (LC) with intact, top-down, and bottom-up data to accurately de novo sequence antibody mixtures.

Our complete solution for sequencing antibody mixtures will be offered under PEAKS AB Polyclonal Antibody Sequencing Service by Bioinformatics Solutions Inc. Mass Spec Lab.

Key Features:

  • End-to-End Workflow Solution: Accurate sequencing of antibody mixtures directly by mass spectrometry
  • Protein de novo Sequencing: No requirement for custom database generation from next-generation sequencing data
  • Optimised LC: Reliable and efficient separation of simple antibody mixtures from polyclonal antibodies
  • In-Depth Characterisation: Complete sequence coverage of top 4 most abundant antibody sequences
  • 3-Tier Leu vs. Ile Differentiation: Differentiation of isoleucine and leucine in 4 most abundant antibodies, using advanced EThcD MS method, enzyme digestion specificity and homology database analysis
  • Validation by Intact Mass: Intact mass measurements of heavy and light chains for confirmation of assembled de novo sequences for top 4 most abundant antibodies in a mixture
  • PolySeq.AI: Multi-level mass spectrometry sequencing powered by innovative, deep learning-enabled data analysis algorithms
  • Fast turnaround time: Comprehensive reports delivered in 4-6 weeks

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Figure 1. PEAKS AB Polyclonal Antibody Sequencing workflow.

Here, we show an example of sequencing a mixture of 4 antibodies (Adalimumab, Bevacizumab, Rituximab, and Herceptin) without the requirement for a custom database from next-generation sequencing data, to simulate an LC fraction from separation of polyclonal antibody samples. Integrating intact, top-down, and bottom-up data generates correct sequences of all 4 antibodies. Adalimumab is shown as an example.

Figure 2. Intact masses of 4 antibodies mixture matched with the theoretical masses from de novo sequencing. (a) Whole intact, (b) reduced intact, and (c) table of theoretical and measured masses matching within 100 PPM error tolerance.
Figure 3. Assembled sequence from PolySeq.AI. De novo sequenced heavy chain with bottom-up supporting peptides assembled to top-down fragment ions (indicated by blue lines).

Your research is important to us. We want to help you get the results you’re looking for. Contact us to schedule a free consultation to discuss your project. We will help you determine the optimal experimental setup for a fast turnaround time and with the best price.

Method Overview

Our novel platform integrates optimised LC separation with multi-level mass spectrometry techniques using our advanced PolySeq.AI deep learning-enabled software algorithms to resolve and accurately sequence antibody mixtures without the requirement for custom database generation from next-generation sequencing data. Those unique capabilities developed at BSI will extend the well-established PEAKS AB Service and represent a significant development towards sequencing polyclonal antibodies.

Service Description:

We use optimised LC methods to efficiently resolve and separate simple antibody mixtures from a complex sample. Each LC fraction is divided into 3 portions and used for intact, bottom-up, and top-down data acquisition. All data are combined within PolySeq.AI for assembly of multiple antibody sequence constructs. LC-MS and intact mass measurements are performed as illustrated below and used for correct pairing of heavy and light chains. Manual verification and sequence analysis are always included, and a comprehensive report is delivered upon project completion. Glycan profiling and a targeted search for post-translational modifications can be included as an optional add-on to this service.


Intact method

Top-down method

Bottom-up method

PolySeq.AI Analysis: bottom-up peptide assembly + top-down method + intact mass measurements + I/L differentiation (EThcD MS) and PTM analysis + heavy and light chain pairing + manual verification + comprehensive report.

What we need:

  • Sample amount: >1mg
  • Samples must be free of detergents and polymers (Triton X-100, Tween-20, PEG, etc.)
  • Buffer information

Sequencing Data

100% Sequence Accuracy

Our PEAKS AB Antibody Protein Sequencing Services include stringent quality controls on all MS methods (top-down, bottom-up, and intact mass). Direct fragment ion evidence from MS/MS data is required for each amino acid in the assembled protein sequences. Our MS workflows and PolySeq.AI deep learning-enabled algorithms reduce data complexity and enable accurate assembly of bottom-up peptides to longer top-down peptide fragment ions. Multiple intact mass measurements are performed to ensure the correct pairing of heavy and light chains.

Confident Leucine / Isoleucine Differentiation

Isobaric Leucine (Leu) and Isoleucine (Ile) residues are generally considered to be indistinguishable by MS. Due to this ambiguity, it is difficult to differentiate between the two residues and this can impose serious consequences on the overall performance of the antibody’s specificity and affinity. In our PEAKS AB Sequencing Services, we use an integrated 3-tier strategy that combines 1) w-ion detection in EThcD, 2) enzyme cleavage preference, and 3) homology statistics for unambiguous discrimination of Leu/Ile residues.

Heavy / Light Chain Pairings

Advanced PolySeq.AI algorithms use a combination of intact mass measurements of whole antibodies, reduced heavy and light chains, and Fd’ heavy chain subunits to correctly pair heavy and light chains of antibodies in a mixture.

Custom Deliverables

The assembly of your results and project report will include:

  • Antibody sample descriptions and detailed experimental procedures
  • Complete sequences of up to 4 antibodies per sample fraction
  • Intact mass measurements with heavy and light chain pairings
  • Top-down peptide mapping and fragment ion annotation
  • Bottom-up peptide mapping with confidence levels for each amino acid and identified post-translation modifications
  • I/L differentiation and statistics
  • High-quality peptide spectra that span variable regions

Want to know more? Request more details to learn why our methodology and experience gives you better results than our competitors. Have questions? We’ll be happy to answer any of your inquiries today.