With the introduction of combinatorial chemistry, many high throughput screening technologies are being developed for discovering drugs, for screening the affinities of these many drug candidates with target proteins, and for determining protein-protein binding interactions. Associated analytical measurements include NMR, X-ray crystallography, mass spectrometry, chemical microarrays [133, 134] and protein microarrays [135, 136]. An automated approach for the analysis of protein structure by H/D exchange and MS was reported recently  (see also Chapter 12). A more recent publication utilizes a fully automated system to differentiate partial and full agonists of the ligand binding domain of the nuclear receptor PPARg . Other relatively new mass spectrometry-based methods are SUPREX [80, 139], frontal affinity chromatography with MS (FAC-MS)  (see also Chapter 6), MS-based diffusion measurements , ''SpeedScreen'' by size exclusion chromatography (SEC) and LC-MS , affinity capillary electrophoresis MS (ACE-MS) , and pulsed ultrafiltration-mass spectrometry (PUF-MS)  (see Chapter 4), and they have the potential for high throughput. High throughput is achieved by using automated sample preparation with robot systems and parallel LC/MS with autosampling and online desalting. These may be adapted for PLIMSTEX and FPOP.
Although PLIMSTEX and FPOP were originally developed using LC/ESI-MS, it does not eliminate the possibility of using MALDI for the protein-ligand titration. A different desalting procedure is needed, and the conditions for quench (PLIMSTEX) and analysis would be controlled differently than when using LC/ESI-MS. If successful, automated procedures for MALDI-MS could also be immediately adopted for PLIMSTEX and FPOP. Nevertheless, we are not recommending these approaches for fast screening of libraries containing thousands or millions of compounds as there are simpler assay methods available, including direct MS measurements of complexes. In the subsequent lead optimization phase of drug development, a cluster of related compounds may be selected. Their subtly different activities need to be quantified or defined more accurately, which may be a role for PLIMSTEX and FPOP.
368 | 11 Quantification of Protein-Ligand Interactions in Solution Acknowledgements
The research at Washington University was supported by the National Center for Research Resources of the National Institutes of Health, Grant P41RR00954, and by a supplemental grant from that resource. We acknowledge Don Rempel for his help in the development of modeling procedures for PLIMSTEX and we thank Dr. Zhaohui Du and Dr. Raghu Chitta for some of the kinetics and titrations. We acknowledge Ilan Vidavsky, Jim Walters, and Henry Rohrs for assistance with data collection and analysis involving fast radical footprinting development. We acknowledge donations of protein from collaborators Dr. B. Pramanik (Schering-Plough Research Institute), Prof. D. Cistola (Washington University) and Prof. M. Shea (University of Iowa).
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