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Ecto-Fc MS identifies ligand-receptor interactions through extracellular domain Fc fusion protein baits and shotgun proteomic analysis
- Source :
- Nature Protocols
- Publication Year :
- 2014
- Publisher :
- Springer Science and Business Media LLC, 2014.
-
Abstract
- Ligand-receptor interactions represent essential biological triggers which regulate many diverse and important cellular processes. We have developed a discovery-based proteomic biochemical protocol which couples affinity purification with multidimensional liquid chromatographic tandem mass spectrometry (LCLC-MS/MS) and bioinformatic analysis. Compared to 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 four 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 to identify extracellular ligand – receptor interactions.
- Subjects :
- Proteomics
Recombinant Fusion Proteins
Shotgun
macromolecular substances
Computational biology
Biology
Sensitivity and Specificity
Article
General Biochemistry, Genetics and Molecular Biology
Protein protein interaction network
Domain (software engineering)
Mice
03 medical and health sciences
0302 clinical medicine
Tandem Mass Spectrometry
parasitic diseases
Extracellular
Animals
Humans
Staphylococcal Protein A
Receptor
030304 developmental biology
0303 health sciences
Computational Biology
food and beverages
Ligand (biochemistry)
Molecular biology
Microspheres
Immunoglobulin Fc Fragments
Fc fusion
HEK293 Cells
Membrane protein
Immunoglobulin G
030217 neurology & neurosurgery
Chromatography, Liquid
Subjects
Details
- ISSN :
- 17502799 and 17542189
- Volume :
- 9
- Database :
- OpenAIRE
- Journal :
- Nature Protocols
- Accession number :
- edsair.doi.dedup.....ddea18db47174e94c4a607287c9d0432