1. Protocol for identifying physiologically relevant binding proteins of G-protein-coupled receptors
- Author
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Bill X. Huang and Hee-Yong Kim
- Subjects
Proteomics ,Protein expression and purification ,Mass Spectrometry ,Science (General) ,Q1-390 - Abstract
Summary: G-protein-coupled receptors (GPCRs) are important therapeutic targets expressed on the cell surface. Here, we present a protocol for identifying physiologically relevant binding proteins of adhesion GPCR GPR110. We describe steps for in-cell chemical crosslinking, immunoprecipitation, and quantitative high-resolution mass spectrometry. Notably, we detail a label-free quantitation strategy that eliminates irrelevant interacting proteins using an inactive GPR110 mutant with impaired surface expression. Furthermore, we outline procedures for validating the identified partners.For complete details on the use and execution of this protocol, please refer to Huang et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
- Published
- 2023
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