A Protocol for Protein Profiling Using Chemoselective Cleavable Linker Probes in Semi-permeabilized Cells

Area of Science:

• Biochemistry
• Chemical Biology
• Proteomics

Background:

• Activity-based protein profiling (ABP) is crucial for identifying disease-associated enzymes.
• Detecting low-abundance proteins remains a challenge in biological research.
• Chemically cleavable linkers can improve the efficiency of ABP assays.

Purpose of the Study:

• To enhance the sensitivity and efficacy of activity-based protein profiling.
• To optimize the use of chemically cleavable linkers in ABPs.
• To improve the detection of low-abundance enzymes involved in disease.

Main Methods:

• Incorporation of chemically cleavable linkers into activity-based probes (ABPs).
• Affinity purification of target enzymes using ABPs.
• Chemical cleavage of linkers to release captured enzymes and interaction partners.
• Direct delivery of ABPs to semi-permeabilized cells.

Main Results:

• Chemically cleavable linkers significantly enhance the detection of target enzymes.
• Delivery of ABPs to semi-permeabilized cells further increases assay sensitivity and efficacy.
• This method allows for specific release and identification of enzymes and their interacting proteins.

Conclusions:

• Activity-based probes with cleavable linkers are effective tools for enzyme identification in disease research.
• Optimizing probe delivery to semi-permeabilized cells enhances detection capabilities.
• This approach facilitates the discovery of novel disease biomarkers and therapeutic targets.