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

Abstract

A protocol is described for rapidly screening small organic molecules for their ability to bind a target protein while obtaining structure-related information as part of a structure-based drug discovery and design program. The methodology takes advantage of and combines the inherent strengths of size exclusion gel chromatography, mass spectrometry, and NMR to identify bound complexes in a relatively universal high-throughput screening approach. Size exclusion gel chromatography in the spin column format provides the high-speed separation of a protein-ligand complex from free ligands. The spin column eluent is then analyzed under denaturing conditions by electrospray ionization mass spectrometry (MS) for the presence of small molecular weight compounds formerly bound to the protein. Hits identified by MS are then individually assayed by chemical shift perturbations in a 2D 1H-15N HSQC NMR spectrum to verify specific interactions of the compound with the protein and identification of the binding site on the protein. The utility of the MS/NMR assay is demonstrated with the use of the catalytic fragment of human fibroblast collagenase (MMP-1) as a target protein and the screening of a library consisting of approximately 32 000 compounds for the identification of molecules that exhibit specific binding to the RGS4 protein.

Development of a Screening Assay for Ligands to the Estrogen Receptor Based on Magnetic Microparticles and LC-MS

“…Since mass spectrometry provides excellent selectivity, sensitivity, and structural information to facilitate the identification of ligands in complex mixtures, various screening assays have been developed based on multidimensional chromatography mass spectrometry, such as sizeexclusion chromatography LC-MS [6], ultrafiltration LC-MS [7], affinity capillary electrophoresis-MS [8], H/D exchange MALDI MS [9], and FT-ICR MS [10]. For example, we have reported the development of ultrafiltration LC-MS to screen mixtures of compounds for ligands to various receptors, such as estrogen receptors (ER) [11], cyclooxygenase-2 [12] and human serum albumin [13].…”

Covalent modification of stathmin by CCNU determined by FTMS analysis of modified proteins and tryptic peptides

“…Mass spectrometry has been used as a tool for characterizing protein-small molecule interactions in solution [1][2][3][4][5][6]. One of the critical aspects of such studies is the ability to assess the extent and/or sites of small molecule incorporation.…”

Determining the optimal size of small molecule mixtures for high throughput NMR screening

“…Additionally, NMR may be used to evaluate the physical properties of a chemical lead, measure K D 's (Fielding, 2003), identify ligand binding sites (Roberts, 2000), and determine a co-structure (Clore and Gronenborn, 1994;Cooke, 1997;Kay, 1997;Roberts, 2000). A diverse number of NMR screening approaches have been developed, which include SAR by NMR (Shuker et al, 1996;Hajduk et al, 1997a;Hajduk et al, 1997c;Hajduk et al, 1999b;Johnson et al, 2003), SHAPES (Moore et al, 2004;Lepre et al, 2002;Fejzo et al, 1999), and MS/NMR (Moy et al, 2001). NMR spectroscopy is a relatively insensitive technique requiring higher amounts of material and acquisition time compared to standard methods used in traditional HTS assays.…”