Measuring protein structural changes on a proteome-wide scale using limited proteolysis-coupled mass spectrometry

Area of Science:

• Proteomics
• Biophysics
• Molecular Biology

Background:

• Protein structural dynamics are crucial for cellular functions.
• Existing biophysical methods struggle to analyze proteome-wide structural changes in complex biological extracts.
• A need exists for methods to assess protein structural alterations within their native cellular environment.

Purpose of the Study:

• To introduce and validate Limited proteolysis-coupled mass spectrometry (LiP-MS) as a proteome-wide approach for detecting protein structural changes.
• To demonstrate the applicability of LiP-MS in complex biological contexts.
• To highlight the potential of LiP-MS for various biological and medical applications.

Main Methods:

• Proteome extracts undergo sequential protease digestion: non-specific protease under native conditions, followed by trypsin under denaturing conditions.
• Structure-specific peptides are generated for bottom-up mass spectrometry (MS) analysis.
• Label-free quantification via shotgun or targeted MS measures structure-dependent proteolytic patterns.

Main Results:

• LiP-MS enables the identification of protein structural alterations directly within complex biological extracts.
• The method allows for proteome-wide scale analysis of structural changes.
• Specific protein regions involved in structural transitions or affected by binding events can be identified.

Conclusions:

• LiP-MS is a powerful proteomics technique for analyzing protein structural changes in their native biological context.
• Applications include discovering perturbation-induced alterations, identifying drug targets, and detecting disease-associated protein structural states.
• The approach facilitates the study of protein aggregates and binding events directly in biological samples.