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

The ability to sequence proteins without reliance on a genomic template defines a critical frontier in proteomics. This approach, known as de novo protein sequencing, is essential for applications in antibody sequencing, microbiome proteomics, and antigen discovery, which require accurate reconstruction of target sequences. To advance this field, we here explore two hyperthermoacidic archaeal (HTA) proteases for de novo antibody sequencing, benchmarking them against trypsin and chymotrypsin. Each HTA-protease generated about five times more unique peptide reads than trypsin or chymotrypsin, providing high redundancy across all complementarity-determining regions. Combined with EAciD fragmentation on a ZenoTOF, this methodology enabled complete, unambiguous antibody sequencing. De novo analysis showed much higher alignment scores and reduced the sequence errors by using the HTA-generated data. With short digestion times, minimal sample cleanup, and analysis in just a single liquid chromatography-mass spectrometry (LC-MS/MS) run, this streamlined single-protease approach delivers a scalable and efficient strategy for de novo protein sequencing across diverse applications. A record of this paper's transparent peer review process is included in the supplemental information.