Identifying lipid-protein interactomes with functional lipid probes

Area of Science:

• Cellular Biology
• Biochemistry
• Proteomics

Background:

• Cellular lipids are crucial in health and disease, but their interactions with proteins are not well understood.
• Existing methods for studying lipid-protein interactions are limited, lacking a practical, dedicated protocol.

Purpose of the Study:

• To develop and detail a practical protocol for identifying lipid-protein interactions within cells.
• To enable the characterization of lipid interactomes using multifunctionalized lipid derivatives.

Main Methods:

• Cells are treated with lipid derivatives followed by photochemistry to create lipid-protein conjugates.
• Cellular lysates are prepared for downstream analysis using click chemistry with either a fluorophore or azide beads.
• Lipid-protein conjugates are visualized via in-gel fluorescence or prepared for proteomic analysis.

Main Results:

• A comprehensive protocol is provided for determining lipid interactomes.
• The protocol includes steps for cell treatment, lysate extraction, and conjugate analysis via fluorescence or proteomics.
• Guidelines for controls, troubleshooting, and important parameters are included.

Conclusions:

• This protocol allows for the identification of proteins that bind to specific lipids across various biological systems and cellular states.
• The workflow facilitates a major advance in characterizing the biological roles of lipids in cellular systems.
• The method enables the identification of the interactome for select lipid species.