MS/NMR: A Structure-Based Approach for Discovering Protein Ligands and for Drug Design by Coupling Size Exclusion Chromatography, Mass Spectrometry, and Nuclear Magnetic Resonance Spectroscopy

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• 作者：Franklin J. Moy ; Kevin Haraki ; Dominick Mobilio ; Gary Walker ; Robert Powers ; Keiko Tabei ; Hui Tong ; and Marshall M. Siegel
• 刊名：Analytical Chemistry
• 出版年：2001
• 出版时间：February 1, 2001
• 年：2001
• 卷：73
• 期：3
• 页码：571 - 581
• 全文大小：333K
• 年卷期：v.73,no.3(February 1, 2001)
• ISSN：1520-6882

文摘

A protocol is described for rapidly screening small organicmolecules for their ability to bind a target protein whileobtaining structure-related information as part of a structure-based drug discovery and design program. Themethodology takes advantage of and combines the inherent strengths of size exclusion gel chromatography, massspectrometry, and NMR to identify bound complexes ina relatively universal high-throughput screening approach.Size exclusion gel chromatography in the spin columnformat provides the high-speed separation of a protein-ligand complex from free ligands. The spin column eluentis then analyzed under denaturing conditions by electrospray ionization mass spectrometry (MS) for the presenceof small molecular weight compounds formerly bound tothe protein. Hits identified by MS are then individuallyassayed by chemical shift perturbations in a 2D ¹H-¹⁵NHSQC NMR spectrum to verify specific interactions of thecompound with the protein and identification of thebinding site on the protein. The utility of the MS/NMRassay is demonstrated with the use of the catalytic fragment of human fibroblast collagenase (MMP-1) as a targetprotein and the screening of a library consisting of~32 000 compounds for the identification of moleculesthat exhibit specific binding to the RGS4 protein.