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.