title: A protocol for the systematic and quantitative measurement of protein-lipid interactions using the liposome-microarray-based assay.
abstract: Lipids organize the activity of the cell's proteome through a complex network of interactions. The assembly of comprehensive atlases embracing all protein-lipid interactions is an important challenge that requires innovative methods. We recently developed a liposome-microarray-based assay (LiMA) that integrates liposomes, microfluidics and fluorescence microscopy and which is capable of measuring protein recruitment to membranes in a quantitative and high-throughput manner. Compared with previous assays that are labor-intensive and difficult to scale up, LiMA improves the protein-lipid interaction assay throughput by at least three orders of magnitude. Here we provide a step-by-step LiMA protocol that includes the following: (i) the serial and generic production of the liposome microarray; (ii) its integration into a microfluidic format; (iii) the measurement of fluorescently labeled protein (either purified proteins or from cell lysate) recruitment to liposomal membranes using high-throughput microscopy; (iv) automated image analysis pipelines to quantify protein-lipid interactions; and (v) data quality analysis. In addition, we discuss the experimental design, including the relevant quality controls. Overall, the protocol-including device preparation, assay and data analysis-takes 6-8 d. This protocol paves the way for protein-lipid interaction screens to be performed on the proteome and lipidome scales.
authors.affiliation: European Molecular Biology Laboratory (EMBL) Structural and Computational Biology Unit, Heidelberg, Germany.|European Molecular Biology Laboratory (EMBL) Cell Biology and Biophysics Unit, Heidelberg, Germany.
authors.forename: Antoine-Emmanuel
authors.initials: AE
authors.lastname: Saliba
authors.affiliation: European Molecular Biology Laboratory (EMBL) Structural and Computational Biology Unit, Heidelberg, Germany.
authors.forename: Ivana
authors.initials: I
authors.lastname: Vonkova
authors.affiliation: European Molecular Biology Laboratory (EMBL) Structural and Computational Biology Unit, Heidelberg, Germany.
authors.forename: Samy
authors.initials: S
authors.lastname: Deghou
authors.affiliation: European Molecular Biology Laboratory (EMBL) Structural and Computational Biology Unit, Heidelberg, Germany.
authors.forename: Stefano
authors.initials: S
authors.lastname: Ceschia
authors.affiliation: European Molecular Biology Laboratory (EMBL) Advanced Light Microscopy Facility, Heidelberg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Tischer
authors.affiliation: European Molecular Biology Laboratory (EMBL) Structural and Computational Biology Unit, Heidelberg, Germany.
authors.forename: Karl G
authors.initials: KG
authors.lastname: Kugler
authors.affiliation: European Molecular Biology Laboratory (EMBL) Structural and Computational Biology Unit, Heidelberg, Germany.
authors.forename: Peer
authors.initials: P
authors.lastname: Bork
authors.affiliation: European Molecular Biology Laboratory (EMBL) Cell Biology and Biophysics Unit, Heidelberg, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Ellenberg
authors.affiliation: European Molecular Biology Laboratory (EMBL) Structural and Computational Biology Unit, Heidelberg, Germany.
authors.forename: Anne-Claude
authors.initials: AC
authors.lastname: Gavin
mesh_terms: D002460:Cell Line; D006801:Humans; D007091:Image Processing, Computer-Assisted; D056656:Lab-On-A-Chip Devices; D008081:Liposomes / Q000378:metabolism*; D046228:Microarray Analysis / Q000295:instrumentation / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011786:Quality Control
chemical_list: D008081:Liposomes; D011506:Proteins