ArticleNature protocols2017
Synthesis of a HyCoSuL peptide substrate library to dissect protease substrate specificity.
Article in Nature protocols, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 53 papers.
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Who cites it
53 citing papers in PubMed, 105 citations in OpenAlex.
- Article
- Engineering Cathepsin S Selective Chemical Probes and Antibody-Drug Conjugates through Substrate Profiling with Unnatural Amino Acids.Journal of medicinal chemistry · 2026Article
- Proteolysis activity mapping and substrate discovery platform for identifying tumor-activated biosensors.Nature chemical biology · 2026Article
- Using a bioluminescence resonance energy transfer caspase biosensor to study caspase-3 cleavage site specificity.Bioscience reports · 2026Article
- Coupling high-throughput protease enzymology with viral replication reveals biochemical constraints of viral fitness.bioRxiv : the preprint server for biology · 2026Article
- Chemical probes for enzyme imaging: challenges in design, synthesis and biomedical applications.Chemical science · 2025Review
- Article
- Chemical tools to define and manipulate interferon-inducible Ubl protease USP18.Nature communications · 2025Article
- Use of protease substrate specificity screening in the rational design of selective protease inhibitors with unnatural amino acids: Application to HGFA, matriptase, and hepsin.Protein science : a publication of the Protein Society · 2024Article
- Review
- Chemical tools to define and manipulate interferon-inducible Ubl protease USP18.bioRxiv : the preprint server for biology · 2024Article
- Thermoprotection by a cell membrane-localized metacaspase in a green alga.The Plant cell · 2024Article
- Non-Canonical Amino Acids in Analyses of Protease Structure and Function.International journal of molecular sciences · 2023Review
- Advancement of Computational Design Drug Delivery System in COVID-19: Current Updates and Future Crosstalk- A Critical update.Infectious disorders drug targets · 2023Article
- Chemical Tools to Image the Activity of PAR-Cleaving Proteases.ACS bio & med chem Au · 2023Review
- In-Depth Specificity Profiling of Endopeptidases Using Dedicated Mix-and-Split Synthetic Peptide Libraries and Mass Spectrometry.Analytical chemistry · 2023Article
- Identification of Human Host Substrates of the SARS-CoV-2 MACS infectious diseases · 2023Article
- Targeting caspase-2 interactions with tau in Alzheimer's disease and related dementias.Translational research : the journal of laboratory and clinical medicine · 2023Review
- Fluorescent Activity-Based Probe To Image and Inhibit Factor XIa Activity in Human Plasma.Journal of medicinal chemistry · 2023Article
- Embracing enzyme promiscuity with activity-based compressed biosensing.Cell reports methods · 2023Article
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Authors and funding
3 authors at 3 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Many biologically and chemically based approaches have been developed to design highly active and selective protease substrates and probes. It is, however, difficult to find substrate sequences that are truly selective for any given protease, as different proteases can demonstrate a great deal of overlap in substrate specificities. In some cases, better enzyme selectivity can be achieved using peptide libraries containing unnatural amino acids such as the hybrid combinatorial substrate library (HyCoSuL), which uses both natural and unnatural amino acids. HyCoSuL is a combinatorial library of tetrapeptides containing amino acid mixtures at the P4-P2 positions, a fixed amino acid at the P1 position, and an ACC (7-amino-4-carbamoylmethylcoumarin) fluorescent tag occupying the P1' position. Once the peptide is recognized and cleaved by a protease, the ACC is released and produces a readable fluorescence signal. Here, we describe the synthesis and screening of HyCoSuL for human caspases and legumain. We also discuss possible modifications and adaptations of this approach that make it a useful tool for developing highly active and selective reagents for a wide variety of proteolytic enzymes. The protocol can be divided into three major parts: (i) solid-phase synthesis of the fluorescence-labeled HyCoSuL, (ii) screening of protease P4-P2 preferences, and (iii) synthesis of the optimized activity probes equipped with an AOMK (acyloxymethyl ketone) reactive group and a biotin label for easy detection. Beginning with the library design, the entire protocol can be completed in 4-8 weeks (HyCoSuL synthesis: 3-5 weeks; HyCoSuL screening per enzyme: 4-8 d; and activity-based probe synthesis: 1-2 weeks).
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