High-Throughput Screening by Mass Spectrometry: Comparison with the Scintillation Proximity Assay with a Focused-File Screen of AKT1/PKBα

Abstract

Mass spectrometry is an emerging format for label-free high-throughput screening. The main limitation of mass spectrometry is throughput, due to the requirement to purify samples prior to ionization. Here the authors compare an automated high-throughput mass spectrometry (HTMS) system (RapidFire) with the scintillation proximity assay (SPA). The cancer therapy target AKT1/PKBalpha was screened against a focused library of kinase inhibitors and IC50 values determined for all compounds that exhibit > 50% inhibition. A selection of additional compounds that exhibited <or= 50% inhibition in the primary screen was chosen as controls to confirm inactives. The selection of compounds is expected to identify common actives, common inactives, false positives, and false negatives. Agreement is found between HTMS and SPA in terms of primary hit identification and hit confirmation.

Label-Free High-Throughput Screening via Mass Spectrometry: A Single Cystathionine Quantitative Method for Multiple Applications

“…[4][5][6][7][8][9] However, the ABBREVIATIONS: AOA, aminooxyacetic acetate; BGG, bovine gamma-globulin; CBS, cystathionine beta-synthase; DMEM, Dulbecco's modifi ed Eagle's medium; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; EC 50 , effective dose, 50%; EDTA, ethylenediaminetetraacetic acid; ESI, electrospray ionization; FBS, fetal bovine serum; HPLC, high performance liquid chromatography; HTMS, high throughput mass spectrometry; HTS, high throughput screen(ing); IC 50 , inhibitory concentration at 50% inhibition; LCMS, liquid chromatography-mass spectrometry; LOQ, limit of quantitation; MALDI, matrix-assisted laser desorption ionization; MEM, minimum essential medium; MS, mass spectrometry; MUX, multiplexed; NEAA, nonessential amino acids; PLP, pyridoxal 5'-phosphate; PMSF, phenylmethanesulfonyl fl uoride; PPG, propargylglycine; Q, quadrupole; QqQ, triple quadrupole; SAM, S-(5'-adenosyl)-L-methionine; SPE, solid phase extraction; SRM, single reaction monitoring; TCA, trichloroacetic acid.…”

Mass spectrometry approaches to monitor protein–drug interactions

“…hundreds of protein kinases. Recent advances in mass spectrometry not only enabled the development of MS-based assay systems that offer similar throughput and precision as traditional assays [7,8] but, perhaps more importantly, enabled new chemoproteomics-based approaches aimed at defining and quantifying the interactions of small molecule test compounds with native proteins in cell extracts or cell fractions, under conditions carefully optimized to preserve protein integrity, folding, posttranslational modifications, and sometimes also the interaction of the target with regulatory proteins. Methods for the identification of direct drug-protein interactions have become important tools in modern drug discovery for identification of the target protein(s) by which bioactive molecules, e.g.…”

High-Throughput Screening Using Mass Spectrometry within Drug Discovery

“…Matrix-assisted laser desorption ionization (MALDI)-MS has been successfully used for higher throughput screening, but the drawback is multiple transfer steps of reaction mixture and potentially significant matrix effects [7]. It was not until the introduction of RapidFire™-MS, integration of automated plate handling, and simple sample preparation with a standard MS that high-throughput screening (HTS) could really access MS end points [8][9][10]. Sample processing time was reduced to seconds; hence the throughput was increased significantly.…”