期刊
NATURE PROTOCOLS
卷 9, 期 9, 页码 2061-2074出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2014.140
关键词
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资金
- US National Institutes of Health (NIH) Pathway to Independence Award [K99DC013805-01]
- NIH [R01 MH068770, P41 GM103533, R01MH100175, HHSN268201000035C, R01NS067216, R01MH068578, R01-MH092906]
- Robert Wood Johnson Foundation [67038]
- NARSAD Young Investigator Award from the Brain and Behavior Research Foundation
- European Research Council (ERC) [311083]
- Research Foundation Flanders (FWO) Odysseus Grant
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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