Rapid and Label-Free Structural Proteomics Using One-Step Swift Trypsin LiP-MS.

Area of Science:

• Proteomics
• Structural Biology
• Biochemistry

Background:

• Limited proteolysis mass spectrometry (LiP-MS) is crucial for studying protein conformational changes.
• Conventional LiP-MS workflows are complex, reducing reproducibility and sensitivity.
• A simplified, rapid, and sensitive method is needed for proteome-wide structural analysis.

Purpose of the Study:

• To develop a simplified one-step protocol for LiP-MS, named Swift Trypsin LiP-MS (STLiP-MS).
• To enhance the sensitivity and reproducibility of proteomic structural analysis.
• To demonstrate the method's applicability in identifying protein-protein interactions and drug targets.

Main Methods:

• Developed STLiP-MS using a trypsin-immobilized spin column and high-speed centrifugation for surface-limited proteolysis.
• Optimized the protocol by suppressing predigestion and enabling immediate enzyme inactivation.
• Applied STLiP-MS to HEK293 cell extracts and the adenosine A2A receptor (A2A-BRIL).

Main Results:

• STLiP-MS identified 286 proteins with conformational changes in HEK293 cells, with 37 linked to phosphatase activity.
• Enhanced sensitivity detected 799 proteins with structural alterations, 77 enriched in phosphatase-related categories.
• Validated antibody-induced protection on A2A-BRIL and confirmed interaction sites using cryogenic electron microscopy.

Conclusions:

• STLiP-MS is a rapid, robust, and sensitive platform for label-free detection of local structural changes under near-physiological conditions.
• The method accurately predicts protein-protein interaction sites.
• STLiP-MS shows significant promise for structural proteomics and drug target identification.