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.

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Figure 1: Schematic principle of the liposome-microarray-based assay (LiMA) and protocol flowchart.
Figure 2: Protective sticker preparation (Steps 21–30).
Figure 3: Spotting platform.
Figure 4: Microfluidic channel bonding protocol.
Figure 5: Setup for the injection of sample into the microfluidic device.
Figure 6: Workflow of automated image analysis and data quality control.
Figure 7: Anticipated results.

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Acknowledgements

We are grateful to the EMBL Advanced Light Microscopy Facility (ALMF), C. Gehin and C. Merten for expert help. We also thank other members of P.B.'s, J.E.'s and A.-C.G.'s groups for continuous discussions and support. This work is partially funded by the DFG in the framework of the Cluster of Excellence, CellNetworks Initiative of the University of Heidelberg (ExIni, EcTop). A.-E.S. is supported by the European Molecular Biology Laboratory and the EU Marie Curie Actions Interdisciplinary Postdoctoral Cofunded Programme.

Author information

Affiliations

Authors

Contributions

A.-E.S., A.-C.G. and J.E. designed the research. A.-C.G. directed the research. A.-E.S. developed the experimental protocol with the help of I.V. and S.C. A.-E.S., I.V., K.G.K. and C.T. developed the image analysis pipeline. S.D. and P.B. developed the bioinformatics tools.

Corresponding author

Correspondence to Anne-Claude Gavin.

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Competing interests

A.-E.S., I.V., J.E. and A.-C.G. declare competing financial interests in the form of an international patent application (PCT/EP2013/065256) based on the method LiMA.

Integrated supplementary information

Supplementary Figure 1 Equipment requirement setup.

(a) Glass-slide holder. A home-made glass-slide holder accommodates approx. 20 glass slides with a stirring magnetic bar beneath the glass slides for the washing steps. (b) Spotting rack in a transparent Mylar bag. The spotting rack can be directly plugged in the spotting robot and the lipid solution will be drawn from the vials through the septum. (c) Photo of spotting platform. (d) Photo of the device holder plugged on the microscope.

Supplementary Figure 2 From spotting preparation to spotted-TAL storage.

(a) Photo of the spotting robot. (b-c) After spotting, the protective stickers are removed from the spotted-TALs with tweezers (photo b) and glass slides are removed from the spotting platform (photo c).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–2, Supplementary Data 1 (PDF 518 kb)

Supplementary Table 1

Guide for the calculation of lipid compositions of lipid mixtures used for LiMA (XLSX 17 kb)

Supplementary Data 2

Transparency masks for lithography Raw transparency mask files in dxf format, to make microfluidic channels, spotting platform and protective stickers (ZIP 312 kb)

Supplementary Data 3

CellProfiler pipeline and R script rLiMA (ZIP 37288 kb)

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Saliba, A., Vonkova, I., Deghou, S. et al. A protocol for the systematic and quantitative measurement of protein–lipid interactions using the liposome-microarray-based assay. Nat Protoc 11, 1021–1038 (2016). https://doi.org/10.1038/nprot.2016.059

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