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Application of Affinity Selection-Mass Spectrometry Assays to Purification and Affinity-Based Screening of the Chemokine Receptor CXCR4

[ Vol. 15 , Issue. 6 ]


Charles E. Whitehurst, Zhiping Yao, Denise Murphy, Mingxuan Zhang, Shane Taremi, Lisa Wojcik, Julie M. Strizki, Jack D. Bracken, Cliff C. Cheng, Xianshu Yang, Gerald W. Shipps, Michael Ziebell and Elliott Nickbarg   Pages 473 - 485 ( 13 )


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.


Affinity selection, antagonist, chemokine, combinatorial libraries, CXCR4, G-protein coupled receptor, high throughput screening, ligand binding assay, mass spectrometry, purification, protein target


Merck Research Laboratory, 33 Avenue Louis Pasteur, Boston, MA 02215, USA.

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