Identifying Lipid–protein Interactomes with Functional Lipid Probes

Cellular lipids shape health and disease through specific protein interactions, yet lipid–protein networks remain poorly defined. Despite rapid advances in functional lipid probes, the field still lacks a practical, dedicated protocol for conducting lipid–protein interaction studies. We describe detailed methods for determining lipid interactomes within cells using multifunctionalized lipid derivatives. We provide a protocol that details how to (i) treat cells with lipid derivatives and perform photochemistry to obtain lipid–protein conjugates, (ii) extract cellular lysates for downstream analysis, (iii) perform click chemistry on lysates with a fluorophore and observe lipid–protein conjugates by in-gel fluorescence and (iv) perform click chemistry on lysates with azide beads and prepare lipid–protein conjugates for proteomic analysis. We provide context on important parameters for each step and include guidelines for controls, as well as suggestions for troubleshooting based on common problems encountered during the preparation of this protocol. This protocol enables identification of proteins that bind to specific lipids across diverse biological systems and cellular states. The entire workflow from cell treatment to complete proteomic sample preparation requires ~15 h over 4 d, depending on the type of experimental readout (in-gel fluorescence or proteomics) and the usage of pause points. Practitioners are expected to be familiar with standard biochemical techniques, such as sterile sample handling and tissue culture and gel electrophoresis. Additional skills are needed for mass spectrometric analysis, and collaboration with a proteomics core facility is recommended. The described procedures uniquely enable the identification of the protein interactors (the interactome) of select lipid species, providing for a major advance in the characterization of the biological roles of lipids in cellular systems. Cellular lipids are known to have specific protein interactions. This protocol describes how to study the lipid–protein interactome using multifunctional lipid probes capable of photo-cross-linking and click chemistry by in-gel fluorescence or mass spectrometry.