Comprehensive assembly of monoclonal and mixed antibody sequences

The elucidation of antibody sequence information is crucial for understanding antigen binding and advancing therapeutic and research applications. However, complete de novo assembly of monoclonal antibody sequences remains challenging due to accuracy and robustness limitations. To address this issue, we introduce Fusion, an innovative de novo assembler that integrates overlapping peptides and template information into complete sequences using a beam search strategy. We demonstrate Fusion’s performance by reconstructing multiple human and murine antibodies with highest accuracy (100% and over 99%, respectively). Biological validation of the recombinantly expressed AFS98 antibody with unknown sequences further supports its effectiveness. Furthermore, current methods are applicable only to traditional monoclonal antibody sequencing assembly, presenting a significant bottleneck in achieving higher throughput. In contrast, Fusion can assemble peptide sequences from mixtures of two or three monoclonal antibodies into complete individual sequences with the same accuracy as traditional sequencing, significantly enhancing throughput. To our knowledge, this is the first study enabling high-throughput sequencing of multiple antibodies using only bottom-up mass spectrometry. The duration, expense, and reagent consumption of mass spectrometry detection are comparable to those required for sequencing a single monoclonal antibody. In summary, Fusion’s superior performance in handling the complex antibody sequencing represents a significant advancement in antibody research.