Comprehensive Identification of Lipid–Membrane Protein Interactions via Advanced Proteomics and Extended Lipid-Immobilized Bead Technology

In biological membranes, lipids interact with membrane proteins (MPs) and play important roles in allosterically regulating their structure and function. Analyzing lipid-MP interactions is necessary for understanding these regulatory mechanisms; however, there have been few comprehensive and systematic studies to date. To address this, we developed a high-sensitivity, high-throughput platform that integrates lipid-immobilized beads with advanced proteomics to analyze lipid-MP interactions in detail. We prepared six types of lipid-immobilized beads, including sphingomyelin (SM), ceramide (Cer), dihydrosphingomyelin (DHSM), dihydroceramide (DHCer), phosphatidylcholine (PC), and cholesterol (Chol). In addition, we introduced a novel type of beads that immobilized SM and Chol (SM/Chol beads) to mimic lipid rafts. We first demonstrated that SM/Chol beads coprecipitated with Nakanori, a protein that specifically recognizes and binds to SM/Chol complexes, whereas beads immobilized with SM or Chol alone did not coprecipitate. This indicates the effectiveness of SM/Chol beads for the identification of raft-associated proteins. Next, the cell lysates were incubated with the seven types of lipid-immobilized beads and the recovered proteins were analyzed using shotgun proteomics. This approach successfully identified over 7000 lipid-binding proteins. Filtering based on fold-change values and subsequent enrichment analysis revealed distinct binding protein profiles for each lipid, highlighting the functional diversity of lipid-MP interactions and their roles in cellular processes. In summary, our methodology enables an unprecedented large-scale exploration of lipid-MP interactions. This platform provides a versatile tool for examining the lipid-mediated regulation of MPs and offers new insights into the physiological significance of the lipidome and its implications for health and disease.