XA-Novo: an accurate and high-throughput mass spectrometry-based de novo sequencing technology for monoclonal antibodies and antibody mixtures
preprint · Research Square · 2025
| Date | 2025-02-07 |
| Type | preprint |
| Venue | Research Square |
| Publisher | Research Square |
| Contribution | algorithm |
| DOI | 10.21203/rs.3.rs-5858891/v1 |
| Citations (OpenAlex) | 3 |
Peer-reviewed version: XA-Novo: an accurate and high-throughput mass spectrometry-based de novo sequencing technology for monoclonal antibodies and antibody mixtures (2026-02-15, Nature Communications)
Abstract
Elucidating antibody sequences by mass spectrometry-based de novo sequencing is essential but remains technically challenging. Here we present XA-Novo, an accurate and high-throughput de novo sequencing solution that integrates a single-pot multi-enzymatic gradient digestion method with a beam search-based assembler (Fusion) to reconstruct full-length antibody sequences directly from bottom-up mass spectrometry data. Benchmarking across well-characterized antibodies from multiple species demonstrates that XA-Novo outperforms commercial solutions in identification sensitivity, sequence completeness, and reconstruction accuracy. Furthermore, XA-Novo successfully reconstructs six immunotherapeutic antibodies with unknown sequences, and in vitro/vivo assays validate that these generated antibodies exhibit functionality equivalent to their commercial counterparts. Moreover, XA-Novo achieves over 99.54% accurate sequence coverage in distinguishing mixed COVID-19 neutralizing antibodies, exceeding the performance of current assemblers reported for single-antibody sequencing. Overall, XA-Novo establishes a reliable, scalable, and broadly applicable workflow for routine antibody sequencing, thereby accelerating both fundamental antibody research and therapeutic antibody development.
Methods and tools
- XA-Novo: NAR knowledge distillation