Abstract

RapidFire
high-throughput mass spectrometry (RapidFire-MS) is a solid phase extraction
(SPE) based, label-free method for screening enzyme targets by directly
detecting and quantifying native substrates and products based on their
mass-to-charge (m/z) ratio. This system therefore, avoids the need for coupled
reactions and reduces interference from fluorescent small molecules that
confound conventional high-throughput assays. The Agilent RapidFire-MS platform
automates quantitative analysis of data from 384-well assay plates at a
sampling rate of ~10 s per well.  Recently,
RapidFire-MS has been successfully used for the screening of enzymes, such as
HIV-I protease (PR), S-adenosylhomocysteine hydrolase (AHCY) and S-Adenosylmethionine
decarboxylase (AdoMetDC) against libraries of a few hundred thousand
small-molecule compounds. Here we describe the use of RapidFire-MS for
high-throughput screening of drug targets for infectious diseases including Trypanosoma brucei Kinetochore Kinase 19
(TbKKT19) and Pseudomonas aeruginosa β-hydroxydecanoyl-(acyl-carrier-protein)
dehydratase (FabA). When compared to
the available fluorescence assays, the RapidFire-MS assays use less enzyme
(reducing cost and the tight binding limit) and exhibit a higher signal to
blank ratio as well as Robust Z’. In addition, the highly specific direct
measurement of reaction product significantly decreases the false positive detection
rate. In conclusion, our data indicates that RapidFire-MS provides a superior
screening platform compared to traditional fluorescence-based methods.