High-fidelity bispecific protease KRysin for deeper plasma proteomics and phosphoproteomics
- 1Genovis AB
Abstract
Bottom-up proteomics relies on efficient and reproducible proteolytic digestion. Yet trypsin, the most widely used protease, frequently misses cleavages, particularly near prolines, acidic and phosphorylated residues. Incomplete digestion inflates search space, dilutes peptide signal, and limits depth and reproducibility, especially in challenging matrices such as plasma and phosphoproteomics. Here we present KRysin, a recombinant bispecific protease that cleaves C-terminal to lysine and arginine with very high specificity and efficiency, generating tryptic peptides compatible with standard proteomics workflows. In HeLa digests, ~99% of KRysin-generated peptides ended in lysine or arginine, including at sites followed by proline, and only 0.2% of peptides contained an internal (missed-cleaved) arginine and 6% an internal lysine, compared with 9% and 25%, respectively, for trypsin. Using narrow-window DIA at 60 samples-per-day, KRysin quantified close to 50,000 class I phosphosites, >10,000 more than trypsin-based workflows, and identified up to 40% more plasma protein groups, uniquely revealing receptors, cytokines, and growth factors. KRysin therefore provides a direct alternative to trypsin wherever digestion completeness, speed and PTM coverage are limiting.
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BibTeX
@misc{batth2026highfidelity,
title = {{High-fidelity bispecific protease KRysin for deeper plasma proteomics and phosphoproteomics}},
author = {T. S. Batth and C. Hernandez-Rollan and C. Koenig and F. L. Mao and J. V. Olsen},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.20.760339},
url = {https://www.biorxiv.org/content/10.64898/2026.10.20.760339v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - High-fidelity bispecific protease KRysin for deeper plasma proteomics and phosphoproteomics
AU - T. S. Batth
AU - C. Hernandez-Rollan
AU - C. Koenig
AU - F. L. Mao
AU - J. V. Olsen
PY - 2026
DA - 2026/10/20
PB - bioRxiv
DO - 10.64898/2026.10.20.760339
UR - https://www.biorxiv.org/content/10.64898/2026.10.20.760339v1
AB - Bottom-up proteomics relies on efficient and reproducible proteolytic digestion. Yet trypsin, the most widely used protease, frequently misses cleavages, particularly near prolines, acidic and phosphorylated residues. Incomplete digestion inflates search space, dilutes peptide signal, and limits depth and reproducibility, especially in challenging matrices such as plasma and phosphoproteomics. Here we present KRysin, a recombinant bispecific protease that cleaves C-terminal to lysine and arginine with very high specificity and efficiency, generating tryptic peptides compatible with standard proteomics workflows. In HeLa digests, ~99% of KRysin-generated peptides ended in lysine or arginine, including at sites followed by proline, and only 0.2% of peptides contained an internal (missed-cleaved) arginine and 6% an internal lysine, compared with 9% and 25%, respectively, for trypsin. Using narrow-window DIA at 60 samples-per-day, KRysin quantified close to 50,000 class I phosphosites, >10,000 more than trypsin-based workflows, and identified up to 40% more plasma protein groups, uniquely revealing receptors, cytokines, and growth factors. KRysin therefore provides a direct alternative to trypsin wherever digestion completeness, speed and PTM coverage are limiting.
ER -