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 IC 50 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.
INTRODUCTION
HIGH-THROUGHPUT SCREENING (HTS) plays a central role in the drug discovery process starting at the early "hit" discovery stage then continuing through to lead optimization. As such, the screening community has continuously developed new technologies to meet the broadening assay needs and to expedite the work flow of drug discovery. Within the pharmaceutical and biotechnology enzyme screening community, fluorescent-and chemiluminescent-based detection methods continue to be the routine assay platforms of choice for isolated enzyme assays. These assays are desirable because they have been developed to be simple homogeneous assays that often offer universal methods to evaluate a variety of targets using the same reagents. However, 1 of the primary challenges for these approaches continues to be how to maintain the high speed of analysis while minimizing false-positive or false-negative readouts. Inherent to this challenge is the nature of fluorescence and chemiluminescence readout, which can lend itself to false readouts because of the properties of various compound classes that enhance or quench the signals. Thus a comparable approach in speed, robust readout, and cost that further minimizes the potential for false readouts would be a benefit to the field.One approach that offers promise in this arena is mass spectrometry. A variety of mass spectrometry (MS) techniques have been used to measure enzyme activity, enzyme kinetics, inhibition, and more recently for HTS applications (reviewed in the studies of Greis 1 and De Boer et...
Increasing concentrations of nitrate in surface water and groundwater are becoming a worldwide concern, yet little information has been published on toxicity of nitrate to common organisms used for toxicity testing. The acute and chronic toxicity of nitrate (NO3‐N) to Ceriodaphnia dubia, Daphnia magna, and Pimephales promelas was investigated in 48‐h to 17‐d laboratory exposures. The 48‐h median lethal concentration (LC50) of nitrate to C. dubia and D. magna neonates was 374 mg/L NO3‐N and 462 mg/L NO3‐N. The no‐observed‐effect concentration (NOEC) and the lowest‐observed‐effect concentration (LOEC) for neonate production in C. dubia were 21.3 and 42.6 mg/L NO3‐N, respectively. The NOEC and LOEC values for neonate production in D. magna were 358 and 717 mg/L NO3‐N, respectively. The 96‐h LC50 for larval fathead minnows (P. promelas) was 1, 341 mg/L NO3‐N. The NOEC and LOEC for 7‐d larval and 11‐d embryo‐larval growth tests were 358 and 717 mg/L NO3‐N, respectively. Additional exposure of breeding P. promelas and their fertilized eggs to nitrate did not increase susceptibility further. The LC50 values for all species tested were above ambient concentrations of nitrate reported for surface water. However, the LOEC for C. dubia was within the range of concentrations that could be found in streams draining areas under extensive agricultural cultivation.
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