Graphical abstract. From Pañeda et al., 2026. “Deep coverage and extended sequence reads obtained with a single archaeal protease expedite de novo protein sequencing by mass spectrometry.“ Cell Systems (2026). doi: https://doi.org/10.1016/j.cels.2026.101536; Licensed under the terms of the Creative Commons CC-BY 4.0 license.

De novo protein sequencing by mass spectrometry, where peptide sequences are determined directly from MS data without a genomic reference, is increasingly important for applications such as antibody sequencing and microbiome proteomics. A key bottleneck is achieving sufficient sequence coverage of hypervariable antibody regions (CDRs), which define antigen binding specificity. Current workflows typically require parallel digestion with four to six proteases, increasing experimental and computational complexity. Standard proteases like trypsin generate short, narrow-mass-range peptides that rarely achieve complete coverage of CDR regions, while longer non-tryptic peptides are difficult to fragment confidently with conventional collision-induced dissociation (CID).

Pañeda et al. aimed to simplify de novo antibody sequencing by evaluating two hyperthermoacidic archaeal (HTA) proteases (Krakatoa and Vesuvius) paired with a hybrid electron-activated collision-induced dissociation (EAciD) fragmentation strategy on a SCIEX ZenoTOF 7600 system. Peptides were separated using an Aurora® Elite™ 15×75 XS C18 UHPLC column coupled to an UltiMate 3000 in a single LC-MS/MS run per sample.

Using a four-antibody mixture as a polyclonal mimic, a single HTA protease generated up to five times more unique peptides than trypsin, with a median per-amino-acid redundancy of 23–24 compared to 1–3 for trypsin and chymotrypsin. De novo sequence alignment scores improved four-fold, with a corresponding four-fold reduction in sequencing errors. The researchers demonstrated that a single HTA protease, paired with EAciD fragmentation and nano-LC-MS/MS, can replace the conventional multi-protease workflow for de novo antibody sequencing – a finding with significant implications for antibody discovery, clinical diagnostics, and analysis of any protein lacking a genomic reference.

Publication
Cell Systems

Authors

Laura Pérez Pañeda, Tereza Kadavá, Tatiana M. Shamorkina, Douwe Schulte, Patrick Pribil, Sibylle Heidelberger, Allison Michele Narlock-Brand, Steven M. Yannone, Joost Snijder, & Albert J.R. Heck;

Title

Deep coverage and extended sequence reads obtained with a single archaeal protease expedite de novo protein sequencing by mass spectrometry