Advanced Strategies in Liposome Microarray-Based Assays for Protein–Lipid Interactions

The importance of sphingolipids and phosphoinositides in cellular functions is highlighted, given the intricate nature of the lipidome and the critical role of membrane proteins. There are many challenges in scaling up classical assays, such as surface plasmon resonance and isothermal titration calorimetry, to the proteome and lipidome levels. Novel approaches, such as LiMA23, a high-throughput liposome microarray assay, have been introduced, enabling automated and multiplexed quantification of membrane protein recruitment with minimal sample requirements. This discussion extends to the roles of lipids and liposomes in biological research, highlighting their structural, signaling, and energy storage functions. Liposomes mimic cellular membranes and are invaluable for studying dynamic and context-dependent protein–lipid interactions. The versatility of microarray technology has been explored in genomics, proteomics, and glycomics, emphasizing its crucial role in understanding complex cellular networks. The advantages of employing microarrays to study protein–lipid interactions include the simultaneous analysis of numerous molecules, providing insights into structure–function relationships. The detailed design and fabrication of the liposome microarrays were elucidated, including substrate preparation, lipid selection, vesicle formation, array printing, and quality control. Immobilization strategies for proteins on liposome microarrays, such as covalent bonding, adsorption, and entrapment, are discussed, each with unique benefits and considerations. This chapter provides a systematic guide for conducting protein–lipid interaction assays using liposome microarrays, incorporating various analytical methods, such as fluorescence microscopy, surface plasmon resonance, and atomic force microscopy. Critical parameters and metrics for data analysis, such as binding affinity, fluorescence intensity, colocalization, and kinetic parameters, are outlined. Applications of liposome microarrays, such as glycolipid–protein interactions and the methodical LiMA characterization of protein–lipid interactions, have been investigated. This chapter concludes with a comparative analysis of liposome microarrays with other techniques, such as surface plasmon resonance and liposome pull-down assays. Future directions and innovations, including single-molecule imaging, live-cell imaging with microfluidics, and enhanced data analysis pipelines, offer insights into the evolving landscape of protein–lipid interaction research. It is suggested that biosensors and computational modeling be integrated in the future to gain a deeper comprehension of intricate cellular environments. Overall, this chapter is a roadmap for future research and technological advancements in unraveling the intricacies of protein–lipid interactions.