Proteomic profiling of mechanistically distinct enzyme classes using a common chemotype

Area of Science:

• Proteomics
• Chemical Biology
• Enzymology

Background:

• Proteomics research needs methods to analyze protein expression and function in complex biological samples.
• Activity-based protein profiling (ABPP) uses chemical probes to label active enzymes, but lacks probes for many enzyme classes.
• Discovering novel probes is crucial for expanding ABPP applications.

Purpose of the Study:

• To develop a combinatorial strategy for discovering new activity-based proteomics probes.
• To identify novel enzyme targets using a library of sulfonate ester chemotype probes.
• To apply these probes to identify enzymes associated with pathological states, specifically human breast cancer.

Main Methods:

• A combinatorial library of sulfonate ester chemotype probes was synthesized.
• The probe library was screened against complex proteomes for activity-dependent protein labeling.
• Proteins labeled by the probes were identified and characterized.
• The identified probes were used to investigate enzyme activity in human breast cancer cell lines.

Main Results:

• Screening identified at least six mechanistically distinct enzyme classes labeled by the sulfonate ester probes.
• None of the identified enzyme targets were previously known targets for proteomics probes.
• The sulfonate library successfully identified an omega-class glutathione S-transferase.
• This enzyme's activity was found to be upregulated in invasive human breast cancer lines.

Conclusions:

• A combinatorial strategy can accelerate the discovery of activity-based proteomics probes for enzyme classes lacking known affinity labels.
• The developed probes are compatible with whole-proteome analysis.
• This approach can identify enzymes associated with specific pathological conditions, such as breast cancer.