Complementary Methods for de Novo Monoclonal Antibody Sequencing to Achieve Complete Sequence Coverage
peer-reviewed · Journal of Proteome Research · 2020
| Date | 2020-07-02 |
| Type | peer-reviewed |
| Venue | Journal of Proteome Research |
| Publisher | American Chemical Society (ACS) |
| Contribution | downstream-application |
| DOI | 10.1021/acs.jproteome.0c00223 |
| Citations (OpenAlex) | 14 |
| Venue 2-year citedness | 3.83 |
Abstract
Mass spectrometry is a powerful tool for de novo sequencing of novel proteins. Recent efforts in this area have mainly focused on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Here, we present an alternative method, capillary electrophoresis tandem mass spectrometry (CE-MS/MS), for sequencing novel monoclonal antibodies. Using less than 200 ng in total of tryptic digest sample in a triplicated measurement, CE-MS/MS with pH-mediated focusing successfully sequenced mAb infliximab with 100% sequence coverage and 100% accuracy for the light chain and 96% coverage and 93% accuracy for the heavy chain. It was also demonstrated that CE-MS/MS gives comparable results, and in some cases, even better results, as compared to LC-MS/MS when used as a standalone technique. A combined workflow using both CE-MS/MS and LC-MS/MS was also used to sequence a novel antibody, anti-CD-176, resulting in the first proposed sequence for this mAb.
Methods and tools
- Capillary-electrophoresis mAb sequencing: Reaches complete monoclonal antibody sequence coverage by pairing the usual LC-MS/MS with capillary electrophoresis, on the argument that one separation chemistry alone leaves gaps no amount of searching can fill.
Cites (3)
- Evaluating de novo sequencing in proteomics: already an accurate alternative to database-driven peptide identification? (2017) crossref
- Complete De Novo Assembly of Monoclonal Antibody Sequences (2016) crossref
- De novo proteomic sequencing of a monoclonal antibody raised against OX40 ligand (2006) crossref