Target Deconvolution by Limited Proteolysis Coupled to Mass Spectrometry

Area of Science:

• Proteomics
• Biochemistry
• Chemical Biology

Background:

• Limited proteolysis coupled to mass spectrometry (LiP-MS) is an emerging technique.
• It enables structure-based target engagement profiling across the proteome.
• Understanding compound-protein interactions is crucial in drug discovery.

Purpose of the Study:

• To describe a basic protocol for Limited proteolysis coupled to mass spectrometry (LiP-MS).
• To demonstrate the method's utility using rapamycin as an example compound.
• To facilitate the adoption of LiP-MS by researchers with basic biochemistry and mass spectrometry expertise.

Main Methods:

• Incubation of native lysates with a small molecule compound.
• Limited proteolysis using a non-specific protease.
• Analysis of resulting proteolytic fingerprints using mass spectrometry-based proteomics.

Main Results:

• The study provides a clear, adaptable LiP-MS protocol.
• Reference data (PXD035183) is available for method implementation.
• The protocol is demonstrated effectively with the natural product rapamycin.

Conclusions:

• The described LiP-MS protocol is accessible for researchers with foundational knowledge.
• The method allows for straightforward proteome-wide target engagement profiling.
• The protocol's adaptability supports its application to diverse protein samples and small molecules.