On-Chip Preconcentration Microchip Capillary Electrophoresis Based CE-PRM-LIVE for High-Throughput Selectivity...

Area of Science:

• Biochemistry
• Proteomics
• Analytical Chemistry

Background:

• Deubiquitinases (DUBs) are crucial enzymes regulating protein function, with significant implications in human physiology and disease.
• Targeting DUBs for therapeutic intervention is challenging due to the lack of potent and specific small-molecule inhibitors.
• Existing drug discovery methods for DUBs often suffer from low potency and lack of specificity.

Purpose of the Study:

• To develop a rapid and accurate method for activity-based protein profiling (ABPP) of deubiquitinase (DUB) inhibitors.
• To enhance proteome coverage and quantitation integrity for complex biological samples.
• To facilitate the screening of both covalent and reversible DUB inhibitors.

Main Methods:

• Integration of a novel capillary electrophoresis microchip with an on-chip C18 solid-phase extraction bed (SPE-ZipChip).
• Application of a tandem mass tagging (TMT) labeled parallel reaction monitoring (PRM-LIVE) acquisition scheme on a timsTOF Pro mass spectrometer.
• Development of a multiplexed assay for quantifying inhibitor binding to endogenous DUBs.

Main Results:

• The SPE-ZipChip significantly improved proteome coverage for complex samples.
• CE-PRM-LIVE demonstrated high quantitation integrity for TMT-labeled samples.
• The developed platform accurately quantified competitive binding of inhibitors across 49 endogenous DUBs in under 15 minutes.

Conclusions:

• The combined SPE-ZipChip and CE-PRM-LIVE technology offers a powerful platform for rapid DUB inhibitor profiling.
• This approach enables accurate assessment of inhibitor potency and specificity, accelerating drug discovery for DUB targets.
• The method provides a robust solution for screening a wide range of DUBs in a time-efficient manner.