title: Mapping protein receptor-ligand interactions via in vivo chemical crosslinking, affinity purification, and differential mass spectrometry.
abstract: Protein receptor-ligand interactions play important roles in mediating enzyme catalysis, signal transduction, and other protein functions. Immunoaffinity purification followed by mass spectrometry analysis is a common method for identifying protein receptor-ligand complexes. However, it is difficult to distinguish between specific protein binding partners and non-specifically bound proteins that co-purify with the complex. In addition, weakly interacting binding partners may dissociate from the protein receptor-ligand complexes during immunoaffinity purification. The combination of chemical crosslinking, affinity purification, and differential mass spectrometry analysis provides a direct method for capturing stable, weak, and transient protein interactions that occur in vivo and in vitro. This approach enables the identification of functional receptor-ligand binding partners with high confidence. Herein, we describe a differential mass spectrometry approach coupled with in situ chemical crosslinking and immunoaffinity purification for identifying receptor-ligand binding partners. In particular, we identified a functional, counter-ligand structure of the natural killer cell p30-related protein.
authors: Department of Systems Immunology, College of Biomedical Science, Institute of Antibody Research, Kwangwon National University, Chuncheon, Republic of Korea.
authors: Kristine M
authors: KM
authors: Kim
authors: Eugene C
authors: EC
authors: Yi
authors: Youngsoo
authors: Y
authors: Kim
mesh_terms: D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D030562:Databases, Protein; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D020014:K562 Cells; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods*; D046912:Multiprotein Complexes / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D055628:Natural Cytotoxicity Triggering Receptor 3 / Q000737:chemistry; D025941:Protein Interaction Mapping / Q000379:methods*; D012680:Sensitivity and Specificity; D012995:Solubility; D013379:Substrate Specificity
chemical_list: D003432:Cross-Linking Reagents; D008024:Ligands; D046912:Multiprotein Complexes; C431710:NCR3 protein, human; D055628:Natural Cytotoxicity Triggering Receptor 3