Activity-Based Protein Profiling of Serine Hydrolases in Bacteria: Methods and Protocols.

Area of Science:

• Chemoproteomics
• Enzymology
• Microbiology

Background:

• Activity-based protein profiling (ABPP) is a powerful chemoproteomic technique.
• It utilizes functionalized covalent enzyme inhibitors, known as activity-based probes (ABPs), to interrogate enzyme activity.
• Understanding active enzymes in bacteria is crucial for various biological and medical applications.

Purpose of the Study:

• To describe experimental protocols for profiling enzyme activities in bacterial cultures.
• To detail the use of both fluorescent and biotinylated ABPs for enzyme detection and identification.
• To provide adaptable methods for targeting diverse enzyme families in bacteria.

Main Methods:

• Development and optimization of ABPP protocols for bacterial samples.
• Application of fluorescent ABPs for gel-based detection and resolution of enzyme activities.
• Utilization of biotinylated ABPs for enrichment and mass spectrometry-based identification of enzymes.
• Optimization for serine hydrolase profiling using fluorophosphonate probes in Gram-positive and Gram-negative bacteria.

Main Results:

• Established robust protocols for ABPP in bacterial systems.
• Demonstrated successful gel-based detection and MS-based identification of active bacterial enzymes.
• Showcased optimized methods for serine hydrolase profiling.
• Validated adaptability for other enzyme families and bacterial species.

Conclusions:

• The described ABPP protocols provide a versatile platform for bacterial enzyme activity profiling.
• These methods facilitate the detection, quantification, and identification of active enzymes in diverse bacterial contexts.
• The protocols are readily adaptable for broader applications in bacterial research and drug discovery.