Protocols for rapid identification of small-molecule metabolite ligands interacting with proteins
Year: 2024
Type: article
Abstract: Metabolite-protein interactions have not been systematically studied due to a lack of effective techniques. Here, we present a protocol for identifying small-molecule metabolite ligands interacting with proteins. We describe steps for mixing the sample with antibodies for immunoprecipitation and applying organic solvent to extract small-molecule metabolites. We then detail procedures for using high-resolution mass spectrometry to quantify small-molecule metabolites bound to proteins. We validate protocol feasibility using the example of an arachidonic acid-Menin protein interaction. For complete details on the use and execution of this protocol, please refer to He et al. 1 • Instructions for preparing endogenous, exogenous, and purified proteins • Steps for identifying metabolite ligands interacting with proteins • Guidance on recognizing and validating potential protein-metabolite interacting complex Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Metabolite-protein interactions have not been systematically studied due to a lack of effective techniques. Here, we present a protocol for identifying small-molecule metabolite ligands interacting with proteins. We describe steps for mixing the sample with antibodies for immunoprecipitation and applying organic solvent to extract small-molecule metabolites. We then detail procedures for using high-resolution mass spectrometry to quantify small-molecule metabolites bound to proteins. We validate protocol feasibility using the example of an arachidonic acid-Menin protein interaction.
Source: STAR Protocols
Authors: Baoyu He, Rou Zhao, Bin Zhang, Qingli Bie
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,500
OpenAlex ID: W4404171041