It is required to obtain the protein sequence of a monoclonal antibody (mAb) for the development of novel biotherapeutics. The need to extensively characterize mAbs at the molecular level presents a unique challenge to drug developers and manufacturers.

PEAKS AB de novo Antibody Protein Sequencing Service provides the answer to this.

Key Features:

  • FAST: 1-2 week turnaround time
  • Full and In-depth Sequence Coverage: Each amino acid is mapped to more than 20 unique peptides and 100% sequence coverage
  • Accuracy: Every amino acid in CDRs is confidently supported by pairs of intense fragment ions in at least 10 MS2 scans
  • Validation by Intact Mass: Intact mass measurements of heavy and light chains for double-confirmation of C-terminal lysine truncation and assembled de novo sequences
  • 3-Tier Leu vs. Ile Differentiation: Differentiation of isoleucine and leucine using advanced EThcD MS method, enzyme digestion specificity and homology database analysis
  • Interactive Viewer: Investigate details of the report directly from the PEAKS AB software GUI

BSI has developed the proprietary de novo antibody protein sequencing technology, based on our PEAKS AB Software, to sequence antibodies 1,2. The Antibody Sequencing Service consists of full length heavy and light-chain antibody sequencing for all species, isotypes and allotypes. Numerous successful cases from our antibody protein sequencing service have confirmed 100% accuracy and 100% coverage. Satisfaction is guaranteed to meet our customers’ needs.

PEAKS AB Service Options

You ProvideWe Deliver
Option 1• Monoclonal antibody sample (ideal: ≥ 0.1 mg, ≥ 95% purity)• Full protein sequences
• Ile/Leu differentiation (optional)
• Comprehensive PEAKS AB report
• Raw LC-MS/MS data (optional)
Option 2• Raw LC-MS/MS data from a monoclonal antibody sample• Full protein sequences
• Comprehensive PEAKS AB report
Ile/Leu Differentiation Only• Purified protein sample (ideal: ≥ 50 µg, ≥ 95% purity)
• Protein sequence
• Ile/Leu differentiation by EThcD MS

If your sample does not meet all of the criteria listed above (limited quantity, lower purity or sample mixtures, rare species or engineered) talk to us first. We may still be able to work with your sample.

Method Overview

Based on 20 years of expertise with mass spectrometry data analysis, our team keeps improving our proprietary workflow and software for de novo antibody sequencing. Our standard procedure combines bottom-up and top-down MS techniques to construct and validate the de novo protein sequence and consists of the following steps:

Bottom-Up Sequencing Procedure

  1. Digest antibody protein into overlapping peptides using an optimized set of orthogonal enzymes
  2. Analyse peptides by high resolution LC-MS/MS (Thermo Orbitrap series mass spectrometer)
  3. Peptide de novo sequencing from tandem mass spectra
  4. Construct antibody sequence by assembling de novo peptide sequences
  5. Ile/Leu differentiation by using advanced EThcD MS method, enzyme digestion specificity and homology database analysis
  6. Manual confirmation and report generation by our scientists

Intact Mass Validation Procedure

  1. Reduce antibody protein to separate light and heavy chains
  2. Remove N-linked glycans from the heavy chain by PNGase F
  3. Analyse the reduced mixtures by LC-MS (Waters Q-TOF mass spectrometer)
  4. Deconvolution of acquired MS spectra to derive masses of light and heavy chains respectively
  5. Manual confirmation and report generation by our scientists

100% Sequence Coverage

Our Antibody Sequencing Service guarantees each amino acid is typically mapped with more than 20 distinct peptides. The peptide mapping in the left is obtained using our PEAKS AB Software at 0.1% of FDR at peptide-spectrum level. Each bar under the sequence denotes a peptide identified from the MS/MS data and the colour represents a specified enzyme used for sample digestion.

100% Sequence Accuracy

The Antibody Protein Sequencing Service carries out quality control at the amino acid level based on the de novo sequencing result. Direct fragment ion evidences from MS/MS data are required for each amino acid in the assembled protein sequences. Every amino acid in CDRs is confidently supported by pairs of intense fragment ions in at least 10 MS2 scans.

Intact Mass Validation

Intact mass measurements of heavy and light chains are performed for double-confirmation of the assembled de novo sequences, N-terminal pyro-glutamate modification, heavy chain C-terminal lysine truncation and N-linked glycans.

Confident Leucine & Isoleucine Differentiation

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 Antibody Sequencing Service, we use an integrated strategy that combines 1) w-ion detection in EThcD, 2) enzyme cleavage preference, and 3) homology statistics for unambiguous discrimination of Leu/Ile residues.

  1. EThcD-based approach using diagnostic w-ions has proven to solve this problem 3,4,5.
(Image cited from Ref. 4)
  1. Enzyme digestion preference, e.g., chymotrypsin and pepsin selectively prefers digestion at C-terminal of Leu over Ile residues.
  1. Homology database search for conservative sites in the constant region and framework region.