title: Ecto-Fc MS identifies ligand-receptor interactions through extracellular domain Fc fusion protein baits and shotgun proteomic analysis.
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.
authors.affiliation: 1] Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA. [2].
authors.forename: Jeffrey N
authors.initials: JN
authors.lastname: Savas
authors.affiliation: 1] Vlaams Instituut voor Biotechnologie (VIB) Center for the Biology of Disease, Leuven, Belgium. [2] Center for Human Genetics, Katholieke Universiteit (KU) Leuven, Leuven, Belgium. [3] Neurobiology Section, Division of Biology, University of California San Diego, La Jolla, California, USA. [4].
authors.forename: Joris
authors.initials: J
authors.lastname: De Wit
authors.affiliation: Child Health Institute of New Jersey and Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Rutgers University, New Brunswick, New Jersey, USA.
authors.forename: Davide
authors.initials: D
authors.lastname: Comoletti
authors.affiliation: New York University School of Medicine, New York, New York, USA.
authors.forename: Roland
authors.initials: R
authors.lastname: Zemla
authors.affiliation: 1] Neurobiology Section, Division of Biology, University of California San Diego, La Jolla, California, USA. [2] Neuroscience Discovery, F. Hoffman-La Roche, Basel, Switzerland.
authors.forename: Anirvan
authors.initials: A
authors.lastname: Ghosh
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA.
authors.forename: John R
authors.initials: JR
authors.lastname: Yates
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D019295:Computational Biology; D057809:HEK293 Cells; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000302:isolation & purification / Q000378:metabolism*; D007074:Immunoglobulin G / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008863:Microspheres; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013205:Staphylococcal Protein A / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A
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