Sample Organization: Single Samples vs Mixtures, Mixture Set-up: Compatibility of Components, Plate Set-up
Since the numbers of compounds to be assayed in a high throughput drug screening campaign are high (>25 000 samples) and the expected number of hits is relatively low (<0.5%), the GPC spin column assays are more efficiently done with mixtures of compounds. In the earliest reported work using the GPC spin column ESI-MS screening assay, mixtures of ten chemically compatible compounds were prepared . An important additional criterion used for selecting the compounds for the mixtures was that the MW of each compound in the mixture differed by at least 3 Da to allow for clear identification of each component by the mass spectrometer, and thereby, the MW effectively becomes an identification tag for each compound screened in the assay. Additional considerations in the selection of compounds for the mixtures are solubility, structural diversity and druglike characteristics . Also, a reasonable balance of acidic and basic molecules was selected to avoid potentially drastic pH changes upon addition to the protein.
Schnier and coworkers  extended the GPC spin column ESI-MS assay for the analysis of a target protein with mixtures of 80 components (5 mM protein, 1 mM per compound). The compounds were pooled using two different procedures so that a specific compound is found in two wells with completely different well-mates. Eighty microtiter plates were prepared where each plate contained 80 compounds and each compound occupied an individual well. One procedure combined the 80 samples from each of the microtiter plates into different wells of a new microtiter plate. The second procedure pooled each sample from similar wells of the 80 microtiter plates into individual wells of a second microtiter plate. Mixtures of 80 components each were found to be optimum for minimizing false positive GPC spin column eluates.
Most recently, Filpuzzi and coworkers incubated mixtures of 400 compounds with a protein and successfully analyzed for non-covalent binders using an array of GPC spin columns with ESI-MS detection in a very high throughput manner [16, 19, 20]. Compound mixtures of various sizes were evaluated when incubated with PKA spiked with staurosporine and in the absence of PKA. Mixtures of 400 compounds each at a concentration of 7 mM and with a protein concentration of 10 mM gave the best results, while mixtures with greater numbers of compounds gave increased numbers of false positive results.
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