Do-It-Yourself De Novo Antibody Sequencing Workflow that Achieves Complete Accuracy of the Variable Regions
peer-reviewed · Journal of Proteome Research · 2025
| Date | 2025-06-06 |
| Type | peer-reviewed |
| Venue | Journal of Proteome Research |
| Publisher | American Chemical Society (ACS) |
| Contribution | algorithm |
| DOI | 10.1021/acs.jproteome.5c00210 |
| Citations (OpenAlex) | 3 |
| Venue 2-year citedness | 3.48 |
Abstract
Antibodies are widely used as research tools or therapeutic agents. Knowing the sequences of the variable regions of an antibody─both the heavy chain and the light chain─is a prerequisite for the production of recombinant antibodies. Mass spectrometry-based de novo sequencing is a frequently used, and sometimes the only approach to gaining this information. Here, we describe a workflow that enables accurate sequence determination of monoclonal antibodies based on mass spectrometry data and freely available software tools. This workflow, which we developed using a homemade anti-FLAG monoclonal antibody as a reference sample, achieved 100% accuracy of the variable regions with clear distinction between leucine (L) and isoleucine (I). Using this workflow, we successfully decoded a monoclonal anti-HA antibody, for which we had no prior knowledge of its sequence. Based on the de novo sequencing result, we generated a recombinant anti-HA antibody, and demonstrated that it has the same specificity, sensitivity, and affinity as the commercial antibody.
Methods and tools
- DIY de novo antibody sequencing workflow: Assembled from off-the-shelf parts rather than new software: multi-protease digestion, complementary fragmentation and existing de novo tools, combined into a protocol that reached complete accuracy across antibody variable regions and decoded an unknown anti-HA antibody.