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Protein Removal for Optimum Sensitivity

For small molecule screening, the presence of the multiply charged protein peaks is desirable since it confirms that the protein passed through the column with non-covalently bound drug. In most cases, the low mass origin for the distribution begins at about m/z 700 and is often above the high mass cutoff for a desirable pharmaceutical. However, there are cases where a desired screening candidate may be above m/z 700 and the protein peaks interfere with the drug candidate. In addition, the presence of the protein may cause ion suppression of the singly charged drug candidates and they may not be observed. Under such circumstances, it would be desirable to remove the protein before assaying the screened eluates. A number of methods have been proposed, including protein precipitation and protein adsorption (Porvair P3 protein precipitation filtration plate, Porvair Sciences, Shepperton, UK). Perhaps the most efficient method is to treat the spin column eluate with acid to liberate the drug from the protein and then apply centrifugal ultrafiltration (Millipore Microconcentrator) to the sample for separating the protein (retentate) from the drug (eluate). The ultrafiltration protein-free eluate is then analyzed by ESI-MS. This is illustrated in Fig. 2.2 for studies of the non-covalent interaction of a CMV protease mutant with DFMK. DFMK was available as an impure mixture producing a mass spectrum exhibiting low abundance molecular ions [M+2H]2+ and [M+H2O+H]1+ (Fig. 2.2A). The mass spectrum for the GPC spin column eluate exhibited the protein peaks overlapping with the [M+H2O+H]1+ ion and a clearly observed [M+2H]2+ ion (Fig. 2.2B). Upon removal of the CMV protein by centrifugal ultrafiltration, a highly sensitive ESI mass spectrum for the purified DFMK was obtained (Fig. 2.2C) [13].

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