XA-Novo: an accurate and high-throughput mass spectrometry-based de novo sequencing technology for monoclonal antibodies and antibody mixtures

Elucidating antibody sequences by mass spectrometry (MS)-based de novo sequencing is essential but challenging. Herein, we propose XA-Novo, an accurate, robust, high-throughput de novo sequencing solution that integrates a single-pot multi-enzymatic gradient digestion method with a beam search-based Fusion assembler to accurately decipher complete antibody sequences at both the sample preparation stage and the data analysis phase. We first benchmark XA-Novo using data from multiple known antibodies across various species, demonstrating that it outperforms commercial solutions in identification capacity, data completeness, and antibody reconstruction accuracy. Furthermore, XA-Novo successfully reconstructs three immunotherapeutic antibodies with unknown sequences, and in vitro assays validating that the generated antibodies exhibit equivalent functionality to their commercial counterparts. Furthermore, XA-Novo excels in discriminating mixtures of COVID-19 neutralizing antibodies, achieving over 99% sequence coverage accuracy, further validating the effectiveness and flexibility of our solution. We anticipate that XA-Novo will be a powerful tool for future antibody research applications.