Abstract
Recently, mass spectrometry (MS)-based readout has been demonstrated to be a highly effective for high throughput screening (HTS) assays. The major advantages compared to the most common fluorescence readout are the paucity of false readouts, reduced reagent costs, and the ability to multiplex assays such that multiple therapeutic targets can be screened for inhibitor hits with one pass through the compound repository. Previously, we have developed MS-based methods for rapid and accurate compound screening for inhibitors to therapeutic targets. However, the limited use of MS-based methods with small test libraries has been insufficient to validate the overall utility of this readout for large screening campaigns. Thus, in this report, the MS-based readout technology was scaled to include a library of 30,400 compounds to systematically validate the reliability of MALDI-MS readout head-to-head versus a traditional methods of HTS. The target enzyme for these comparative assays is PKC-iota, which plays a role cancer cell survival, tumor growth and potentially invasion. First the MS-based assay was fully integrated into an automated workflow on a PerkinElmer Plate:Explorer HTS system in a 384-well format. Then, the primary screen of 30,400 compounds with both the MS and fluorescence-based readouts yielded a hit rate of 0.3% and 0.9% for the two methods, respectively. Only 29% of the MS-based hits confirmed in triplicate assays; however, 95% of those confirmed hits validated as concentration-dependent inhibitors with IC50 value ranging from low nM to high μM inhibitors. By contrast, 58% of the fluorescence hits were deemed as false positives since they produced fluorescence inhibition even in the absences of PKC-iota. Overall the data validate the utility of the MS-based readout in terms of sensitivity, reproducibility and minimal reagent cost. We are now investigating ways to incorporate screening technologies as an additional service and revenue stream for our core laboratory.