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Nat Protoc. 2013;8(7):1321-36. doi: 10.1038/nprot.2013.072. Epub 2013 Jun 13.

Ligand-based receptor identification on living cells and tissues using TRICEPS.

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1
Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland.

Abstract

Physiological responses to ligands such as peptides, proteins, pharmaceutical drugs or whole pathogens are generally mediated through interactions with specific cell surface protein receptors. Here we describe the application of TRICEPS, a specifically designed chemoproteomic reagent that can be coupled to a ligand of interest for the subsequent ligand-based capture of corresponding receptors on living cells and tissues. This is achieved by three orthogonal functionalities in TRICEPS-one that enables conjugation to an amino group containing ligands, a second for the ligand-based capture of glycosylated receptors on gently oxidized living cells and a biotin tag for purifying receptor peptides for analysis by quantitative mass spectrometry (MS). Specific receptors for the ligand of interest are identified through quantitative comparison of the identified peptides with a sample generated by a control probe with known (e.g., insulin) or no binding preferences (e.g., TRICEPS quenched with glycine). In combination with powerful statistical models, this ligand-based receptor capture (LRC) technology enables the unbiased and sensitive identification of one or several specific receptors for a given ligand under near-physiological conditions and without the need for genetic manipulations. LRC has been designed for applications with proteins but can easily be adapted for ligands ranging from peptides to intact viruses. In experiments with small ligands that bind to receptors with comparatively large extracellular domains, LRC can also reveal approximate ligand-binding sites owing to the defined spacer length of TRICEPS. Provided that sufficient quantities of the ligand and target cells are available, LRC can be carried out within 1 week.

PMID:
23764939
DOI:
10.1038/nprot.2013.072
[Indexed for MEDLINE]
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