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.