NDLI: Stable Isotope Labeling Strategy for Protein–Ligand Binding Analysis in Multi-Component Protein Mixtures

Content Provider	Springer Nature Link
Author	DeArmond, Patrick D. West, Graham M. Huang, Hai Tsang Fitzgerald, Michael C.
Copyright Year	2011
Abstract	Described here is a stable isotope labeling protocol that can be used with a chemical modification- and mass spectrometry-based protein–ligand binding assay for detecting and quantifying both the direct and indirect binding events that result from protein–ligand binding interactions. The protocol utilizes an H 2 16 O$_{2}$ and H 2 18 O$_{2}$ labeling strategy to evaluate the chemical denaturant dependence of methionine oxidation in proteins both in the presence and absence of a target ligand. The differential denaturant dependence to the oxidation reactions performed in the presence and absence of ligand provides a measure of the protein stability changes that occur as a result of direct interactions of proteins with the target ligand and/or as a result of indirect interactions involving other protein–ligand interactions that are either induced or disrupted by the ligand. The described protocol utilizes the $^{18}$O/$^{16}$O ratio in the oxidized protein samples to quantify the ligand-induced protein stability changes. The ratio is determined using the isotopic distributions observed for the methionine-containing peptides used for protein identification in the LC-MS-based proteomics readout. The strategy is applied to a multi-component protein mixture in this proof-of-principle experiment, which was designed to evaluate the technique’s ability to detect and quantify the direct binding interaction between cyclosporin A and cyclophilin A and to detect the indirect binding interaction between cyclosporin A and calcineurin (i.e., the protein–protein interaction between cyclophilin A and calcineurin that is induced by cyclosporin A binding to cyclophilin A).
Starting Page	418
Ending Page	430
Page Count	13
File Format	PDF
ISSN	10440305
Journal	Journal of The American Society for Mass Spectrometry
Volume Number	22
Issue Number	3
e-ISSN	18791123
Language	English
Publisher	Springer-Verlag
Publisher Date	2011-02-01
Publisher Institution	The American Society for Mass Spectrometry
Publisher Place	New York
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Protein folding Ligand binding Thermodynamics Covalent labeling Quantitative proteomics Cyclophilin A Cyclosporin A Calcineurin Proteomics Bioinformatics Organic Chemistry Biotechnology Analytical Chemistry
Content Type	Text
Resource Type	Article
Subject	Spectroscopy Structural Biology

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