on 09-24-201203:08 AM - edited on 10-15-202111:09 AM by Closed Account
Frei AP, Jeon OY, Kilcher S, Moest H, Henning LM, Jost C, Plückthun A, Mercer J, Aebersold R, Carreira EM, Wollscheid B. Nat Biotechnol. 2012 Oct 10;30(10):997-1001. Many cellular responses are triggered by proteins, drugs or pathogens binding to cell-surface receptors, but it can be challenging to identify which receptors are bound by a given ligand. Here we describe TRICEPS, a chemoproteomic reagent with three moieties-one that binds ligands containing an amino group, a second that binds glycosylated receptors on living cells and a biotin tag for purifying the receptor peptides for identification by quantitative mass spectrometry. We validated this ligand-based, receptor-capture (LRC) technology using insulin, transferrin, apelin, epidermal growth factor, the therapeutic antibody trastuzumab and two DARPins targeting ErbB2. In some cases, we could also determine the approximate ligand-binding sites on the receptors. Using TRICEPS to label intact mature vaccinia viruses, we identified the cell surface proteins AXL, M6PR, DAG1, CSPG4 and CDH13 as binding factors on human cells. This technology enables the identification of receptors for many types of ligands under near-physiological conditions and without the need for genetic manipulations.