Application of Affinity Selection-Mass Spectrometry Assays to Purification and Affinity-Based Screening of the Chemokine Receptor CXCR4
2012
Affinity selection-mass spectrometry (AS-MS) is a sensitive technology for identifying small molecules that
bind to target proteins, and assays enabled by AS-MS can be used to delineate relative binding affinities of ligands for
proteins. 'Indirect' AS-MS assays employ size-exclusion techniques to separate target-ligand complexes from unbound
ligands, and target-associated ligands are then specifically detected by liquid chromatography mass spectrometry. We
report how indirect AS-MS binding assays with known reference control compounds were used as guideposts for
development of an optimized purification method for CXCR4, a G-protein coupled chemokine receptor, for which we
sought novel antagonists. The CXCR4 purification method that was developed was amenable to scale-up and enabled the
screening of purified recombinant human CXCR4 against a large combinatorial library of small molecules by high
throughput indirect AS-MS. The screen resulted in the discovery of new ligands that competed off binding of reference
compounds to CXCR4 in AS-MS binding assays and that antagonized SDF1α-triggered responses and CXCR4-mediated
HIV1 viral uptake in cell-based assays. This report provides a methodological paradigm whereby indirect AS-MS-based
ligand binding assays may be used to guide optimal integral membrane protein purification methods that enable
downstream affinity selection-based applications such as high throughput AS-MS screens.
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