Synthesis and Application of Activity-Based Probes for Proteases.
The detection, visualization, and identification of active proteases can be facilitated by activity-based probes, which covalently bind to a catalytic residue of the target protease. The synthesis of activity-based probes can be challenging. We here outline a simple protocol for probe synthesis based on standard solid phase peptide synthesis followed by capping of the N-terminus with a reactive electrophile as a warhead. The applicability of the probes is illustrated by labeling cysteine proteases in cell and tissue lysates with Western blotting or fluorescence scanning as a readout.
Leibniz Institute for Analytical Sciences ISAS, e.v., Otto-Hahn-Str. 6b, 44227, Dortmund, Germany.
Tim
T
Van Kersavond
Leibniz Institute for Analytical Sciences ISAS, e.v., Otto-Hahn-Str. 6b, 44227, Dortmund, Germany.
Minh T N
MTN
Nguyen
Leibniz Institute for Analytical Sciences ISAS, e.v., Otto-Hahn-Str. 6b, 44227, Dortmund, Germany. steven.verhelst@kuleuven.be.|Department of Cellular and Molecular Medicine, KU Leuven-University of Leuven, Herestraat 49, Box 802, 3000, Leuven, Belgium. steven.verhelst@kuleuven.be.
Steven H L
SHL
Verhelst
D001681:Biological Assay / Q000379:methods*; D057930:Click Chemistry; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation; D015336:Molecular Probe Techniques; D015335:Molecular Probes / Q000737:chemistry; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology; D060327:Solid-Phase Synthesis Techniques
D015335:Molecular Probes; D010455:Peptides; D011480:Protease Inhibitors; D010450:Endopeptidases
Activity profiling; Activity-based probes; Chemical proteomics; Click chemistry; Proteases; Solid phase peptide synthesis