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Synthesis of a HyCoSuL peptide substrate library to dissect protease substrate specificity

Nature Protocols volume 12, pages 2189–2214 (2017)Cite this article

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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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Figure 1: Outline of the hybrid combinatorial substrate library (HyCoSuL) method.
Figure 2: Unnatural amino acids.
Figure 3: Diagram of a 96-well plate for placement of HyCoSuL substrates.
Figure 4: Determination of the caspase-3 S4 pocket preferences.
Figure 5: The selection process for protease-specific substrates and probes.
Figure 6: Unnatural amino acids can be used for various types of peptide substrate libraries.
Figure 7: The simplified algorithm for the synthesis of combinatorial or defined substrate libraries with unnatural amino acids.
Figure 8: General scheme of the building blocks synthesis of legumain-selective activity-based probe.

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Acknowledgements

This work was supported by the Polish Ministry of Science and Higher Education (grant Iuventus Plus IP2012 040172 to M.P.), the Polish National Science Centre (grant 2014/14/M/ST5/00619 to M.D.), and the US National Institutes of Health (grant R01GM099040 to G.S.S.). This project has received founding from the European Union`s Horizon 2020 research and innovation program under Marie Skłodowska-Curie grant agreement no. 661187. The Drag laboratory is supported by the Foundation for Polish Science.

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Authors and Affiliations

  1. Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California, USA

    Marcin Poreba & Guy S Salvesen

  2. Division of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Science and Technology, Wroclaw, Poland

    Marcin Poreba & Marcin Drag

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  1. Marcin Poreba
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  2. Guy S Salvesen
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Contributions

M.P., G.S.S. and M.D. developed the protocol, designed the research, interpreted the data and wrote the protocol. M.P. carried out the experiments in the protocol.

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Correspondence to Marcin Poreba or Marcin Drag.

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Integrated supplementary information

Supplementary Figure 1 HyCoSuL synthesis and quality control exemplified with the P2 sublibrary.

P2 HyCoSuL sublibrary contains amino acids mixtures at P3 and P4 positions. To test whether the coupling of isokinetic mixture provided the equal distribution of amino acids in P3 (and P4) position an Edman degradation can be utilized. After coupling and de-protection of P3 position (steps 38-44) several beads are subjected for the analysis in order to determine the molar distribution of amino acids at the N-terminal end of a peptide. The same procedure can be applied to test the equimolar coupling of amino acids mixture to the P4 position.

Supplementary Figure 2 HR-MS and RP-HPLC analysis of ACC-labeled legumain substrate containing unnatural amino acids.

Substrate was synthesized according to standard solid phase Fmoc/Boc strategy, and purified using reverse phase high performance (pressure) liquid chromatography (RP-HPLC) Waters system with semi-preparative C18 column. The purity of the substrate was confirmed using the analytical HPLC with C18 analytical column (UV detector, 220nm). The molecular mass of the substrate was confirmed using High Resolution Mass Spectrometer WATERS LCT premier XE with Electrospray Ionization (ESI) and Time of Flight (TOF) module.

Supplementary Figure 3 HR-MS and RP-HPLC analysis of biotin-6-ahx-DTyr(tBu)-Tic-Ser(tBu)-COOH.

Peptide was synthesized according to the strategy presented in the main protocol (Block A, steps 100-124), and used without further purification. The purity of the peptide was confirmed using analytical HPLC (UV detector, C8 column, 220nm). The molecular mass of the peptide was confirmed using High Resolution Mass Spectrometer WATERS LCT premier XE with Electrospray Ionization (ESI) and Time of Flight (TOF) module.

Supplementary Figure 4 HR-MS and RP-HPLC analysis of Boc-Asp(Bzl)-AOMK.

AOMK warhead was synthesized according to the strategy presented in the main protocol (Block B, steps 125-137), and purified via extraction. The purity of the product warhead was confirmed using analytical HPLC (UV detector, C8 column, 220nm). The molecular mass of the compound was confirmed using High Resolution Mass Spectrometer WATERS LCT premier XE with Electrospray Ionization (ESI) and Time of Flight (TOF) module.

Supplementary Figure 5 HR-MS and RP-HPLC analysis of biotin-labeled legumain activity containing unnatural amino acids.

Probe was synthesized according to the strategy presented in the main protocol (Block C, steps 138-151), and purified using reverse phase high performance (pressure) liquid chromatography (RP-HPLC) Waters system with semi-preparative C8 column. The purity of the probe was confirmed using analytical HPLC with C8 column (UV detector, 220nm). The molecular mass of the substrate was confirmed using High Resolution Mass Spectrometer WATERS LCT premier XE with Electrospray Ionization (ESI) and Time of Flight (TOF) module. Since the probe is more hydrophobic than the substrate, we used C8 (instead of C18) column to purify and analyze.

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Supplementary Figures 1–5 and Supplementary Table 1. (PDF 2303 kb)

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Poreba, M., Salvesen, G. & Drag, M. Synthesis of a HyCoSuL peptide substrate library to dissect protease substrate specificity. Nat Protoc 12, 2189–2214 (2017). https://doi.org/10.1038/nprot.2017.091

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