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.
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.
|You Provide||We Deliver|
|Option 1||• Monoclonal antibody sample (ideal: ≥ 0.2 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||• 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.
Based on 17 year’s expertise in 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:
Intact Mass Validation Procedure
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 color represents a specified enzyme used for sample digestion.
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.
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.
1. Shan, B. and Xin, L. Integrating de novo Sequencing and Database Search for Monoclonal Antibody Sequencing. J Biomol Tech. 24(Suppl). S62–S63. 1/5/2013.
2. Tran, N.H. et al. Complete De Novo Assembly of Monoclonal Antibody Sequences. Sci Rep. 6:31730. 26/08/2016.
3. Johnson, R. S. et al. Novel Fragmentation Process of Peptides by Collision-Induced Decomposition in a Tandem Mass Spectrometer: Differentiation of Leucine and Isoleucine. Anal Chem. 1987 Nov 1;59(21):2621-5.
4. Xiao, Y. et al. Distinguishing between Leucine and Isoleucine by Integrated LC-MS Analysis using an Orbitrap Fusion Mass Spectrometer. Anal Chem. 2016 Nov 1;88(21):10757-10766.
5. Zhokhov, S. et al. An EThcD-Based Method for Discrimination of Leucine and Isoleucine Residues in Tryptic Peptides. J Am Soc Mass Spectrom. 2017 Aug;28(8):1600-1611.