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

Area of Science:

• Biochemistry
• Chemical Biology
• Proteomics

Background:

• Enzyme activity, particularly of proteases, is crucial for biological processes and disease.
• Activity-based probes (ABPs) are used to study enzyme activity, but common fluorescent probes have spectral overlap issues.
• Multiplex analysis of enzyme activity is limited by the spectral properties of traditional probes.

Purpose of the Study:

• To develop novel protease-selective probes for multiplexed enzyme activity detection.
• To overcome the limitations of spectral overlap in traditional fluorescent probes.
• To enable parallel monitoring of multiple protease activities using mass cytometry.

Main Methods:

• Development of protease-selective lanthanide-labeled probes.
• Application of probes in conjunction with mass cytometry.
• Analysis of protease distribution in cell lines and peripheral blood mononuclear cells.

Main Results:

• Successfully developed lanthanide-labeled probes for protease activity detection.
• Demonstrated multiplexed monitoring of four proteases with distinct active site geometries.
• Identified protease distribution across three cell lines and peripheral blood mononuclear cells.
• Established mass cytometry as a framework for multiplexed enzyme activity detection.

Conclusions:

• Lanthanide-labeled probes combined with mass cytometry enable multiplexed protease activity profiling.
• This approach overcomes spectral overlap limitations of fluorescent probes.
• Provides a powerful tool for studying enzyme activity in complex biological samples.