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<p>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.</p>

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

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年3月12日

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  • Yueting Xiong,
  • Wenbin Jiang,
  • Jin Xiao,
  • Qingfang Bu,
  • Jingyi Wang,
  • Zhenjian Jiang,
  • Ling Luo,
  • Xiaoqing Chen,
  • Yijie Qiu,
  • Yangtao Wu,
  • Fan Liu,
  • Rongshan Yu,
  • Ningshao Xia,
  • Quan Yuan

摘要

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.