A Single-Enzyme Shortcut to Antibody Sequencing

How great would it be if we could sequence proteins without reliance on a genomic template. This approach, termed de novo protein sequencing, is essential for applications such as antibody sequencing, microbiome proteomics, and antigen discovery, which require accurate reconstruction of peptide and protein sequences. While trypsin remains the gold-standard protease in proteomics, its restricted cleavage specificity limits peptide diversity. This constraint is especially problematic in antibody sequencing, where the functionally hypervariable regions often lack canonical tryptic sites. As a result, trypsin-based approaches yield sparse reads, leading to sequence gaps. Multi-protease approaches can improve sequence coverage, but they add complexity, compromise scalability and reproducibility.

Together with Steven Yannone of CinderBio and Patrick Pribil of SCIEX, Laura Pérez Pañeda, Tereza Kadavá and Tatiana Shamorkina we explored two HyperThermoacidic Archaeal (HTA)-proteases as single-enzyme solutions for de novo antibody sequencing. Each HTA-protease generated about five times more unique peptide reads than trypsin or chymotrypsin, providing high redundancy across all CDRs. Combined with EAciD fragmentation on a ZenoTOF 7600 system, this approach enabled complete, unambiguous antibody sequencing. De novo analysis using PEAKS/DeepNovo and Stitch developed by Joost Snijder and Douwe Schulte showed up to fourfold higher alignment scores and reduced sequence errors. Additionally, our new approach offers short digestion times, eliminates extensive cleanup, and enables analysis in a single LC-MS/MS run. This streamlined, single-protease approach delivers therefore performance comparable to multi-enzyme workflows, offering a scalable and efficient strategy for de novo protein sequencing across diverse applications. Check it out!