Affinity Centrifugal Ultrafiltration MicroconcentratorESIMS

An ultrafiltration microconcentrator is a useful tool for separating high-molecular-weightbiopolymers from small molecules. These devices consist of a centrifuge tube, which has mounted on its base an ultrafiltration membrane, which is designed to retain molecules above a selected molecular-weight cutoff. On centrifugation, the higher-molecular-weight components are retained and concentrated in a small volume of retained solvent. These microconcentra-tor devices have been evaluated and used to rapidly and efficiently screen drug candidate libraries by centrifuging incubated drug candidates and protein targets whereby the proteins and noncovalently bound protein-drug complexes are retained and the free drug candidates pass through the ultrafiltration membrane to waste. The protein-drug complexes are then denatured. The formerly bound low-molecular-weight drug candidates are finally separated from the protein and identified by ESI-MS. Henion and co-workers [48] developed an affinity ultrafiltration/ESI-MS technique that used polyclonal antibodies raised against benzodiazepine drugs to capture benzodiazepine-related drug candidates from a chemical library by forming noncovalent immunoaffinity complexes. These complexes were retained by the microconcentrator upon centrifugation, and the other components passed through the membrane to waste. The drugs were liberated from the antibody complexes by acidification and the resulting solution analyzed for the freed drugs by HPLC/ESI-MS. Siegel and co-workers [49] took a similar approach with microconcentra-tors to identify noncovalently bound inhibitors of human cytomegalovirus proteases with flow injection ESI-MS. To reduce false-positive results from compounds that bind nonspecifically to the protein and to the wall and membrane of the microconcentrator, the retained protein, protein-complex, and the microconcentrator have to be washed to remove the extraneous nonspecifi-cally bound drugs. Care must be taken to minimize drug losses from the protein-drug complex, since upon washing equilibrium is reestablished between the complex and its environment, causing dissociation of the complex. These effects are less serious for strongly bound complexes and more significant for weakly bound complexes. As a control for false positives, the screening of a mixture in the absence of a protein could be performed where the components present in the last wash of the microconcentrator are identified by ESI-MS. High throughput could be achieved by use of a 96-well-plate ultrafiltration array with parallel multisprayer ESI-MS analysis [50]. Yang and co-workers [51] reported a related affinity selection method employing membranes and ESI-MS for drug screening. Recently, Comess and co-workers [52,53] used centrifugal ultrafiltration with ESI-MS detection to identify library components that promiscuously bind noncovalently to a variety of proteins, producing false-positive drug screening hits. Mixtures were analyzed containing up to 3000 components with fetal calf serum albumin, a model protein useful for identifying these promiscuous library members. After centrifugal ultrafiltration, the retained protein-ligand complex was washed three times with buffer to remove excess free ligands. The retentate was then collected and treated with methanol to denature and precipitate the protein and with CH2Cl2 to free the bound ligands from the denatured protein. After an additional centrifugation step, the supernate containing the protein free lig-and mixture was analyzed by direct infusion ESI-MS. In the event of a large number of hits, compounds were identified by dereplication of the screen with subsets of the original library.

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Getting to Know Anxiety

Getting to Know Anxiety

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