Protease Specificity Calculator
About the Protease Specificity Calculator
The Protease Specificity Calculator is a free, scientifically accurate online tool that predicts how likely a given peptide sequence is to be cleaved by major human proteases using position-specific scoring matrices (PSSMs) derived from experimentally validated cleavage data published in peer-reviewed literature (MEROPS database and Song et al., 2012; 2020 PLOS Computational Biology paper).
Proteases (also called proteinases or peptidases) are enzymes that hydrolyze peptide bonds with remarkable substrate specificity. Understanding protease specificity is essential in drug design, biomarker discovery, cancer research, apoptosis studies, and biotechnology.
Importance & Applications
This calculator is widely used for:
- Predicting caspase substrates during apoptosis research
- Designing highly specific protease-activated prodrugs
- Identifying potential off-target cleavage in biologics
- Studying matrix metalloproteinase (MMP) activity in cancer metastasis
- Optimizing recombinant protein purification tags
- Developing diagnostic probes and activity-based probes (ABPs)
How to Use This Tool (User Guidelines)
- Enter your peptide sequence in single-letter code (e.g., DEVDG or LEVD↑A)
- Use ↑ or | to mark the scissile bond (cleavage site). If omitted, P1–P1′ bond is assumed.
- Select the protease of interest.
- Click “Calculate Specificity”.
- A higher score = higher likelihood of cleavage by the selected protease.
Calculate Protease Specificity
What Is Protease Specificity?
Protease specificity refers to the preference of a protease for particular amino acid residues at and around the cleavage site (Schechter-Berger notation: …P4-P3-P2-P1 ↓ P1′-P2′-P3′-P4′…). This specificity arises from the architecture of the protease’s S4–S1 and S1′–S4′ subsites.
Modern protease specificity profiling uses large-scale peptide library screening or proteomics (e.g., PICS, TAILS, N-terminomics) to generate position-specific scoring matrices (PSSMs). Our Protease Specificity Calculator implements PSSMs published in high-impact journals, including the comprehensive human protease specificity atlas published in PLOS Computational Biology (2020).
Scientific Basis of This Calculator
Scores are calculated as:
Score = Σ log₂(f_aa,pos / f_background)
where f_aa,pos is the observed frequency of amino acid “aa” at position “pos” in known substrates, and f_background is the natural amino acid frequency. Scores > 0 indicate preference, <0 indicate avoidance.
Why Use a Protease Specificity Calculator?
Experimental determination of specificity is expensive and low-throughput. Computational prediction allows researchers to:
- Screen thousands of potential substrates in seconds
- Prioritize candidates for wet-lab validation
- Design highly selective peptide substrates or inhibitors
- Avoid unwanted proteolysis in therapeutic proteins
Supported Proteases & References
| Protease | Preferred Motif | Key Reference |
|---|---|---|
| Caspase-3 & 7 | DxxD↓G | Thornberry et al., 1997; Julien & Wells, 2017 |
| Caspase-8 | (L/V)ExD↓ | Blanchard et al., 1999 |
| MMP-2 & MMP-9 | GPxG↓Lxxx | Kridel et al., 2001 |
| Trypsin | K/R↓(not P) | Keil, 1992 |
| Thrombin | xx(R/K)↓G | Gallwitz et al., 2012 |
Limitations & Best Practices
While highly accurate, in silico predictions cannot fully replace experimental validation. Factors such as protein conformation, exosites, and cellular localization also influence cleavage.
For research-grade applications, we recommend combining this calculator with tools like PROSPERous, SitePrediction, or GPS-CCD.
© 2025 – Tool developed with scientific rigor and hosted by Agri Care Hub.
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