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Journal of Biomolecular Screening
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Development of an Inhibitor Screening Platform via Mass Spectrometry

Rakesh Rathore

University of Cincinnati, Department of Cancer and Cell Biology, Cincinnati, Ohio

Jay Corr

MDS Analytical Technologies, Concord, Ontario, Canada

George Scott

MDS Analytical Technologies, Concord, Ontario, Canada

Pauline Vollmerhaus

MDS Analytical Technologies, Concord, Ontario, Canada

Kenneth D. Greis

University of Cincinnati, Department of Cancer and Cell Biology, Cincinnati, Ohio, ken.greis{at}uc.edu

Commonly used methods for isolated enzyme inhibitor screening typically rely on fluorescent or chemiluminescent detection techniques that are often indirect and/or coupled assays. Mass spectrometry (MS) has been widely reported for measuring the conversion of substrates to products for enzyme assays and has more recently been demonstrated as an alternative readout system for inhibitor screening. In this report, a high-throughput mass spectrometry (HTMS) readout platform, based on the direct measurement of substrate conversion to product, is presented. The rapid ionization and desorption features of a new generation matrix-assisted laser desorption ionization-triple quadrupole (MALDI-QqQ) mass spectrometer are shown to improve the speed of analysis to greater than 1 sample per second while maintaining excellent Z' values. Furthermore, the readout was validated by demonstrating the ability to measure IC50 values for several known kinase inhibitors against cyclic AMP—dependent protein kinase (PKA). Finally, when the assay performance was compared with a common ADPaccumulation readout system, this HTMS approach produced better signal-to-background ratios, higher Z' values, and a reagent cost of about $0.03 per well compared with about $0.60 per well for the fluorescence assay. Collectively, these data demonstrate that a MALDI-QqQ-MS—based readout platform offers significant advantages over the commonly used assays in terms of speed, sensitivity, reproducibility, and reagent cost. (Journal of Biomolecular Screening 2008:1007-1013)

Key Words: protein kinase A • high-throughput screening • mass spectrometry • matrix-assisted laser desorption ionization (MALDI) • label-free screening

This version was published on December 1, 2008

Journal of Biomolecular Screening, Vol. 13, No. 10, 1007-1013 (2008)
DOI: 10.1177/1087057108326143
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