Abstract
Ligand-receptor interactions represent essential biological triggers that regulate many diverse and important cellular processes. We have developed a discovery-based proteomic biochemical protocol that couples affinity purification with multidimensional liquid chromatographic tandem mass spectrometry (LCLC-MS/MS) and bioinformatic analysis. Compared with previous approaches, our analysis increases sensitivity, shortens analysis duration and boosts comprehensiveness. In this protocol, receptor extracellular domains are fused with the Fc region of IgG to generate fusion proteins that are purified from transfected HEK293T cells. These 'ecto-Fcs' are coupled to protein A beads and serve as baits for binding assays with prey proteins extracted from rodent brain. After capture, the affinity-purified proteins are digested into peptides and comprehensively analyzed by LCLC-MS/MS with ion-trap mass spectrometers. In 4 working days, this protocol can generate shortlists of candidate ligand-receptor protein-protein interactions. Our 'ecto-Fc MS' approach outperforms antibody-based approaches and provides a reproducible and robust framework for identifying extracellular ligand-receptor interactions.
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Acknowledgements
We thank M. O'Sullivan, B. Fonslow, B. Stein and J. Moresco, and members of the Yates and Ghosh laboratories for their feedback on the project, and S. Von Daake for suggestions on the manuscript. We thank T. Südhof (Stanford University) for his generosity in providing the original plasmids of β-NRXN1. This plasmid has been modified to accommodate many different genes and suit a variety of applications, but the original high-expression backbone remains unchanged. J.N.S. is supported by a US National Institutes of Health (NIH) Pathway to Independence Award K99DC013805-01. The Yates laboratory is supported by R01 MH068770, P41 GM103533, R01MH100175 and HHSN268201000035C grants from NIH. We also acknowledge support from NIH grants R01NS067216 and R01MH068578 to A.G. and NIH RO1-MH092906 to D.C., as well as the Robert Wood Johnson Foundation (grant no. 67038) for their support of the Child Health Institute of New Jersey. J.D.W. is funded by a NARSAD Young Investigator Award from the Brain and Behavior Research Foundation, a European Research Council (ERC) Starting Grant (no. 311083) and a Research Foundation Flanders (FWO) Odysseus Grant.
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J.N.S., J.D.W., A.G. and J.R.Y. contributed to the conception and design of the project; J.N.S., J.D.W., D.C. and R.Z. performed the experiments; and J.N.S., J.D.W. and D.C. wrote the manuscript.
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Savas, J., De Wit, J., Comoletti, D. et al. Ecto-Fc MS identifies ligand-receptor interactions through extracellular domain Fc fusion protein baits and shotgun proteomic analysis. Nat Protoc 9, 2061–2074 (2014). https://doi.org/10.1038/nprot.2014.140
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DOI: https://doi.org/10.1038/nprot.2014.140
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