Phosphoramidates as Novel Activity-Based Probes for Serine Proteases

Authors

  • Dr. Ute R. Haedke,

    1. Lehrstuhl für Chemie der Biopolymere, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising (Germany)
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  • Sandra C. Frommel,

    1. Lehrstuhl für Chemie der Biopolymere, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising (Germany)
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  • Fabian Hansen,

    1. Lehrstuhl für Chemie der Biopolymere, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising (Germany)
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  • Dr. Hannes Hahne,

    1. Chair of Proteomics and Bioanalytics, Technische Universität München, Emil Erlenmeyer Forum 5, 85354 Freising (Germany)
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  • Prof. Dr. Bernhard Kuster,

    1. Chair of Proteomics and Bioanalytics, Technische Universität München, Emil Erlenmeyer Forum 5, 85354 Freising (Germany)
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  • Prof. Dr. Matthew Bogyo,

    1. Departments of Pathology and Microbiology & Immunology, Stanford University, School of Medicine, 300 Pasteur Drive, CA 94305 Stanford (USA)
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  • Dr. Steven H. L. Verhelst

    Corresponding author
    1. Lehrstuhl für Chemie der Biopolymere, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising (Germany)
    • Lehrstuhl für Chemie der Biopolymere, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising (Germany)===

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Abstract

Activity-based probes (ABPs) are small molecules that exclusively form covalent bonds with catalytically active enzymes. In the last decade, they have especially been used in functional proteomics studies of proteases. Here, we present phosphoramidate peptides as a novel type of ABP for serine proteases. These molecules can be made in a straightforward manner by standard Fmoc-based solid-phase peptide synthesis, allowing rapid diversification. The resulting ABPs covalently bind different serine proteases, depending on the amino acid recognition element adjacent to the reactive group. A reporter tag enables downstream gel-based analysis or LC-MS/MS-mediated identification of the targeted proteases. Overall, we believe that these readily accessible probes will provide new avenues for the functional study of serine proteases in complex proteomes.

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