Mass Spectrometry-Based De Novo Sequencing of Monoclonal Antibodies Using Multiple Proteases and a Dual Fragmentation Scheme
peer-reviewed · Journal of Proteome Research · 2021
| Date | 2021-07-02 |
| Type | peer-reviewed |
| Venue | Journal of Proteome Research |
| Publisher | American Chemical Society (ACS) |
| Contribution | downstream-application |
| DOI | 10.1021/acs.jproteome.1c00169 |
| Citations (OpenAlex) | 80 |
| Venue 2-year citedness | 3.83 |
Preprint version: Mass spectrometry-based de novo sequencing of the anti-FLAG-M2 antibody using multiple proteases and a dual fragmentation scheme (2021-01-08, bioRxiv)
Abstract
Antibody sequence information is crucial to understanding the structural basis for antigen binding and enables the use of antibodies as therapeutics and research tools. Here, we demonstrate a method for direct de novo sequencing of monoclonal IgG from the purified antibody products. The method uses a panel of multiple complementary proteases to generate suitable peptides for de novo sequencing by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a bottom-up fashion. Furthermore, we apply a dual fragmentation scheme, using both stepped high-energy collision dissociation (stepped HCD) and electron-transfer high-energy collision dissociation (EThcD), on all peptide precursors. The method achieves full sequence coverage of the monoclonal antibody herceptin, with an accuracy of 99% in the variable regions. We applied the method to sequence the widely used anti-FLAG-M2 mouse monoclonal antibody, which we successfully validated by remodeling a high-resolution crystal structure of the Fab and demonstrating binding to a FLAG-tagged target protein in Western blot analysis. The method thus offers robust and reliable sequences of monoclonal antibodies.
Methods and tools
- Multi-protease dual-fragmentation antibody sequencing: Direct de novo sequencing of monoclonal IgG from the purified product with a panel of complementary proteases and both stepped HCD and EThcD on every precursor, demonstrated on the anti-FLAG-M2 antibody.