[ASAP] Multiplexed Probing of Proteolytic Enzymes Using Mass Cytometry-Compatible Activity-Based Probes

[ASAP] Multiplexed Probing of Proteolytic Enzymes Using Mass Cytometry-Compatible Activity-Based Probes:

Abstract

The subset of the proteome that contains enzymes in their catalytically active form can be interrogated by using probes targeted toward individual specific enzymes. A subset of such enzymes are proteases that are frequently studied with activity-based probes, small inhibitors equipped with a detectable tag, commonly a fluorophore. Due to the spectral overlap of these commonly used fluorophores, multiplex analysis becomes limited. To overcome this, we developed a series of protease-selective lanthanide-labeled probes compatible with mass cytometry giving us the ability to monitor the activity of multiple proteases in parallel. Using these probes, we were able to identify the distribution of four proteases with different active site geometries in three cell lines and peripheral blood mononuclear cells. This provides a framework for the use of mass cytometry for multiplexed enzyme activity detection.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.0c06762.

• Selective Cy5-labeled ABPs for the detection of lysosomal proteases in different cell types; detection of legumain and cathepsins in HCT116 cells with fluorescent probes and flow cytometry; whole gels demonstrating the potency and selectivity of TOF-probes for the labeling/inhibition of legumain, cathepsin L, and cathepsin B in human cancer cell lines, HCT-116 and MDA-MB-231; detection of proteases by TOF-probes in HCT-116 cells; selective detection of legumain, cathepsin L, and cathepsin B in HCT-116 cell line; selective labeling of proteases in HCT-116 cells with TOF-probes; detailed gating strategy for non-TOF-probes treated and TOF-probes treated HCT-116 cells; whole gels showing determination of TOF-probes selectivity toward cathepsin L and cathepsin B in THP-1 cells; expression level of legumain, cathepsin L, and cathepsin B in three human cell lines: HCT-116, MDA-MB-231, and THP-1; detection of protease activity in THP-1 cells using TOF-probes; proteases detection in Peripheral Blood Mononuclear Cells; experimental methods and characterization of all compounds (PDF)

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Journal of the American Chemical Society

DOI: 10.1021/jacs.0c06762