Enabling Lead Discovery for Histone Lysine Demethylases by High-Throughput RapidFire Mass Spectrometry

Hutchinson, Sue; Leveridge, Melanie; Heathcote, Michelle L.; Francis, Peter; Williams, Laura; Gee, Michelle; Munoz-Muriedas, Jordi; Leavens, Bill; Shillings, Anthony; Jones, Emma; Homes, Paul; Baddeley, Stuart M.; Chung, Chun‐wa; Bridges, Angela; Argyrou, Argyrides

doi:10.1177/1087057111416660

Cited by 91 publications

(97 citation statements)

References 32 publications

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“…The speed and efficiency of the HT-MS/MS can allow for experiments that would otherwise be deemed "untenable" under normal circumstances. The HT system has been validated as suitable for many drug discoveries [58][59][60][61][62][63][64][65] and ADME (Absorption, Distribution, Metabolism and Excretion) based applications [66]. The main drawback of this method is that disaccharides with identical molecular weights cannot be distinguished.…”

Section: History Of Gag Assay By Tandem Mass Spectrometry (Ms/ms)mentioning

confidence: 99%

Establishment of glycosaminoglycan assays for mucopolysaccharidoses

Tomatsu

Shimada

Mason

et al. 2015

Molecular Genetics and Metabolism

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Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by deficiency of the lysosomal enzymes essential for catabolism of glycosaminoglycans (GAGs). Accumulation of undegraded GAGs results in dysfunction of multiple organs, resulting in distinct clinical manifestations. A range of methods have been developed to measure specific GAGs in various human samples to investigate diagnosis, prognosis, pathogenesis, GAG interaction with other molecules, and monitoring therapeutic efficacy. We OPEN ACCESSMetabolites 2014, 4 656 established ELISA, liquid chromatography tandem mass spectrometry (LC-MS/MS), and an automated high-throughput mass spectrometry (HT-MS/MS) system (RapidFire) to identify epitopes (ELISA) or disaccharides (MS/MS) derived from different GAGs (dermatan sulfate, heparan sulfate, keratan sulfate, and/or chondroitin sulfate). These methods have a high sensitivity and specificity in GAG analysis, applicable to the analysis of blood, urine, tissues, and cells. ELISA is feasible, sensitive, and reproducible with the standard equipment. HT-MS/MS yields higher throughput than conventional LC-MS/MS-based methods while the HT-MS/MS system does not have a chromatographic step and cannot distinguish GAGs with identical molecular weights, leading to a limitation of measurements for some specific GAGs. Here we review the advantages and disadvantages of these methods for measuring GAG levels in biological specimens. We also describe an unexpected secondary elevation of keratan sulfate in patients with MPS that is an indirect consequence of disruption of catabolism of other GAGs.

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Section: History Of Gag Assay By Tandem Mass Spectrometry (Ms/ms)mentioning

confidence: 99%

Establishment of glycosaminoglycan assays for mucopolysaccharidoses

Tomatsu

Shimada

Mason

et al. 2015

Molecular Genetics and Metabolism

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“…The JMJD2c MS assay has been described previously by Hutchinson et al 20 In this assay, the disappearance of H3K9Me 3 histone peptide substrate m/z 1531.1 and the formation of H3K9Me 2 (m/z 1517.1) histone peptide product were monitored. Assays were performed by dispensing 2.5 µL of enzyme solution typically containing 250 nM JMJD2c and 0.25 mg/mL BSA in 50 mM MES (pH 7.0) into 1536-well HiBase microtiter plates containing 50 nL compound or DMSO.…”

Section: Assaysmentioning

confidence: 99%

“…Flag-6His-Tev-JMJD2c (1-336) was previously produced in-house using a baculovirus expression system as described by Hutchinson et al 20 …”

Section: Jmjd2c Expression and Purificationmentioning

confidence: 99%

The Evolution of MALDI-TOF Mass Spectrometry toward Ultra-High-Throughput Screening: 1536-Well Format and Beyond

et al. 2016

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Mass spectrometry (MS) offers a label-free, direct-detection method, in contrast to fluorescent or colorimetric methodologies. Over recent years, solid-phase extraction-based techniques, such as the Agilent RapidFire system, have emerged that are capable of analyzing samples in <10 s. While dramatically faster than liquid chromatography-coupled MS, an analysis time of 8-10 s is still considered relatively slow for full-diversity high-throughput screening (HTS). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) offers an alternative for high-throughput MS detection. However, sample preparation and deposition onto the MALDI target, as well as interference from matrix ions, have been considered limitations for the use of MALDI for screening assays. Here we describe the development and validation of assays for both small-molecule and peptide analytes using MALDI-TOF coupled with nanoliter liquid handling. Using the JMJD2c histone demethylase and acetylcholinesterase as model systems, we have generated robust data in a 1536 format and also increased sample deposition to 6144 samples per target. Using these methods, we demonstrate that this technology can deliver fast sample analysis time with low sample volume, and data comparable to that of current RapidFire assays.

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“…This enabling technology has previously been successfully used in lead discovery campaigns for challenging targets. [14][15][16][17] It overcomes the problem of time-consuming sample separation by using an in-line solid-phase extraction (SPE) cartridge system for sample clean up and analyte concentration. This delivers the sample directly to an electrospray ionization (ESI) triple quadrupole mass spectrometer at a rate of approximately 7 s per well.…”

Section: Introductionmentioning

confidence: 99%

Lead Discovery for Human Kynurenine 3-Monooxygenase by High-Throughput RapidFire Mass Spectrometry

et al. 2014

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Kynurenine 3-monooxygenase (KMO) is a therapeutically important target on the eukaryotic tryptophan catabolic pathway, where it converts L-kynurenine (Kyn) to 3-hydroxykynurenine (3-HK). We have cloned and expressed the human form of this membrane protein as a full-length GST-fusion in a recombinant baculovirus expression system. An enriched membrane preparation was used for a directed screen of approximately 78,000 compounds using a RapidFire mass spectrometry (RF-MS) assay. The RapidFire platform provides an automated solid-phase extraction system that gives a throughput of approximately 7 s per well to the mass spectrometer, where direct measurement of both the substrate and product allowed substrate conversion to be determined. The RF-MS methodology is insensitive to assay interference, other than where compounds have the same nominal mass as Kyn or 3-HK and produce the same mass transition on fragmentation. These instances could be identified by comparison with the product-only data. The screen ran with excellent performance (average Z′ value 0.8) and provided several tractable hit series for further investigation.

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Enabling Lead Discovery for Histone Lysine Demethylases by High-Throughput RapidFire Mass Spectrometry

Cited by 91 publications

References 32 publications

Establishment of glycosaminoglycan assays for mucopolysaccharidoses

Establishment of glycosaminoglycan assays for mucopolysaccharidoses

The Evolution of MALDI-TOF Mass Spectrometry toward Ultra-High-Throughput Screening: 1536-Well Format and Beyond

Lead Discovery for Human Kynurenine 3-Monooxygenase by High-Throughput RapidFire Mass Spectrometry

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