Ecto-Fc MS identifies ligand-receptor interactions through extracellular domain Fc fusion protein baits and shotgun proteomic analysis

Jeffrey N. Savas, Joris De Wit, Davide Comoletti, Roland Zemla, Anirvan Ghosh, John R. Yates*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)2061-2074
Number of pages14
JournalNature Protocols
Volume9
Issue number9
DOIs
StatePublished - Sep 2014

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Fingerprint Dive into the research topics of 'Ecto-Fc MS identifies ligand-receptor interactions through extracellular domain Fc fusion protein baits and shotgun proteomic analysis'. Together they form a unique fingerprint.

Cite this