A multiplex extracellular interactome screening method employing high-avidity nanoparticles
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1.
California Institute of Technology
Abstract
Metazoan cells signal to each other via direct contact between cell surface proteins (CSPs) and by interactions of CSP receptors with secreted ligands. CSP extracellular domain (ECD) interactions control organ development and physiology and are perturbed in disease states. However, because they cannot be accurately assessed using standard high-throughput screening techniques, they are underrepresented in protein interaction databases. Many ECD interactions are of low affinity, and their detection in vitro requires taking advantage of avidity effects, typically by multimerization of fusion proteins. Assays that test only one or a few interactions in each binding reaction are inadequate for global interactome screening. Here we describe a new multiplex method that uses purified dimeric ECD fusion proteins coupled to 60-mer nanoparticles as soluble prey, and the same dimers coupled to spectrally distinguishable fluorescent microspheres (beads) as bait. We add one prey to a mixture of up to 500 baits in a single well, then use a Luminex FLEXMAP 3D (FM3D) instrument to read out bait identity and prey binding. The FM3D measures the fluorescent dye ratio for each bead and simultaneously determines the amount of epitope-tagged prey bound to that bead. We use the method, denoted as the Multiplex Interactome Assay (MPIA), to analyze a proof-of-concept (PoC) set of 41 CSPs and secreted protens that is derived from larger collections examined in two interactome screens that used
ELISA-based binding assays. By analyzing interactions among PoC proteins, we compared the MPIA with earlier screening methods. The MPIA has a dynamic range that is at least 30-fold greater than ELISA-based assays and appears to be more sensitive. By coupling the MPIA to an automated protein production and purification platform, we hope to be able to conduct a screen for interactions among thousands of human CSPs and secreted ligands.
Acknowledgement
This work was funded by an NIH TRO1 grant, GM150125, to K.Z., K. Christopher Garcia (Stanford), and Matthew Thomson (Caltech). Maxine Wang was supported by a fellowship from the H.S. Chau Foundation. Work at the Caltech Protein Expression Center is also supported by the Beckman Institute at Caltech.
Conceptualization, K.Z., M.A.A., W.M.W., J.V., M.L.W.; Investigation, M.A.A., M.L.W., E.G., A.W.L., P.V.; Data Curation and Visualization: W.M.W., M.A.A., M.L.W., K.Z.; Writing and Editing, K.Z., W.M.W., M.A.A., M.L.W.; Supervision, K.Z., W.M.W., J.V.
We thank Matt Thomson, Chris Garcia, Pamela Bjorkman, Barbara Wold, An Zhang, Verona Yue, Dirk Siepe, and Mitchell Guttman for helpful discussions, and Tino Pleiner for the anti-GFP NB construct.
ORCID IDs: K.Z., 0000-0002-6705-5605; J.V., 0000-0002-4314-7163; M.A.A., 0000-
0002-6944-3614; M.L.W., 0000-0002-5285-1857.
Supplemental Material
Supplemental files attached:
- MPIA paper, Fig. 2 data supplement.xlsx
- MPIA paper Fig 3 data supplement.xlsx
- MPIA paper, Fig 4 data supplement.xlsx
- Anaya et al_Fig 6 data supplement.xlsx
- MPIA paper Fig. 7 data supplement.xlsx
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Additional details
- National Institutes of Health
- TRO1 GM150125
- Caltech groups
- Division of Biology and Biological Engineering (BBE)
- Publication Status
- Submitted