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.

Hardware components and software modules were configured to enhance the automation, efficiency, and reliability of a commercial open access atmospheric pressure ionization mass spectrometry (API/MS) system for flow injection analysis. The data massaging module is a versatile package for data manipulation/reduction which is initialized upon detecting the end of data acquisition and can function in parallel during the data acquisition of the next sample. The data interpretation module compares the ions in the acquired mass spectrum with the predicted molecular adduct ions in different charge states, as well as the predicted isotopic distributions, possible artifact, polymer/cluster, byproduct/fragmentation ions, and then uses the results to score the quality of the spectrum. The e-mailing module transmits the spectrum and interpretation report to the desktop computer of the submitting chemist where the spectrum can be displayed and the report viewed. A scheme is also presented for the automated interpretation of an API mass spectrum for the determination of the most likely molecular weights of the components present in an “unknown” sample. Related flow diagrams, algorithms, and applications are illustrated.