Demonstrating Enhanced Throughput of RapidFire Mass Spectrometry through Multiplexing Using the JmjD2d Demethylase as a Model System

Leveridge, Melanie; Buxton, Rachel; Argyrou, Argyrides; Francis, Peter; Leavens, Bill; West, Andrew; Rees, Mike; Hardwicke, Philip; Bridges, Angela; Ratcliffe, Steven J.; Chung, Chun‐wa

doi:10.1177/1087057113496276

Cited by 39 publications

(36 citation statements)

References 25 publications

(39 reference statements)

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“…The technology offers data comparable to that of well-characterized SPE-MS methods, but with sample throughput and assay volumes more suited for full-diversity screening. While groups have reported high-throughput screens in MS-based systems previously, these often require a high-volume 384-well format, and throughput is achieved through either multiplexing 7 or compound pooling, 30 both of which require alterations to standard screening logistics and complicated data analysis. The work described here allows data input directly into standard 1536-well analysis workflows.…”

Section: Application For Uhtsmentioning

confidence: 99%

“…While dramatically faster than HPLC-MS, a sample time of 10 s is still considered relatively slow for HTS or uHTS, where screening of millions of compounds would take many months. RapidFire is therefore more typically employed for compound profiling, focused screening, 7,8 and ADME assays, 9 where compound throughput is <5000 compounds/day in a 96-or 384-well plate format, with sample volumes of >40 µL/well. By comparison, fluorescencebased readouts offer a sample time of <1 s and are typically run in 1536-well plates, with assay reactions in sample volumes of 10 µL or less.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Application For Uhtsmentioning

confidence: 99%

Section: Introductionmentioning

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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“…The RapidFire platform, with a sampling rate of 10 s per well, is not of sufficient throughput to enable full diversity screening of the entire GSK Compound Collection (~2 million molecules) with single substrates. However, it is possible to use multiplexed peptide assays to increase throughput, 37 which could enable a broader screening campaign against LRRK2. Although the focus of the screening effort described here is related to LRRK2 kinase activity, this protein also contains a GTPase domain, and recently, compounds that bind to this site have been described.…”

Section: Compound Screeningmentioning

confidence: 99%

A High-Throughput Screen to Identify LRRK2 Kinase Inhibitors for the Treatment of Parkinson’s Disease Using RapidFire Mass Spectrometry

et al. 2016

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LRRK2 is a large multidomain protein containing two functional enzymatic domains: a GTPase domain and a protein kinase domain. Dominant coding mutations in the LRRK2 protein are associated with Parkinson's disease (PD). Among such pathogenic mutations, Gly2019Ser mutation in the LRRK2 kinase domain is the most frequent cause of familial PD in Caucasians and is also found in some apparently sporadic PD cases. This mutation results in 2-to 3-fold elevated LRRK2 kinase activity compared with wild type, providing a clear clinical hypothesis for the application of kinase inhibitors in the treatment of this disease. To date, reported screening assays for LRRK2 have been based on detection of labeled adenosine triphosphate and adenosine diphosphate or on antibody-based detection of phosphorylation events. While these assays do offer a high-throughput method of monitoring LRRK2 kinase activity, they are prone to interference from autofluorescent compounds and nonspecific events. Here we describe a label-free assay for LRRK2 kinase activity using the RapidFire mass spectrometry system. This assay format was found to be highly robust and enabled a screen of 100,000 lead-like small molecules. The assay successfully identified a number of known LRRK2 chemotypes that met stringent physicochemical criteria.

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“…Another approach is to use different peptides for the same target. This has successfully been done using four different peptides followed by pooling into one plate and analysis on the RapidFire™-MS [11]. …”

Section: Selection Of Columnmentioning

confidence: 99%

High-Throughput Screening Using Mass Spectrometry within Drug Discovery

Rohman

Wingfield

2016

Methods in Molecular Biology

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In order to detect a biochemical analyte with a mass spectrometer (MS) it is necessary to ionize the analyte of interest. The analyte can be ionized by a number of different mechanisms, however, one common method is electrospray ionization (ESI). Droplets of analyte are sprayed through a highly charged field, the droplets pick up charge, and this is transferred to the analyte. High levels of salt in the assay buffer will potentially steal charge from the analyte and suppress the MS signal. In order to avoid this suppression of signal, salt is often removed from the sample prior to injection into the MS. Traditional ESI MS relies on liquid chromatography (LC) to remove the salt and reduce matrix effects, however, this is a lengthy process. Here we describe the use of RapidFire™ coupled to a triple-quadrupole MS for high-throughput screening. This system uses solid-phase extraction to de-salt samples prior to injection, reducing processing time such that a sample is injected into the MS ~every 10 s.

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Demonstrating Enhanced Throughput of RapidFire Mass Spectrometry through Multiplexing Using the JmjD2d Demethylase as a Model System

Cited by 39 publications

References 25 publications

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

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

A High-Throughput Screen to Identify LRRK2 Kinase Inhibitors for the Treatment of Parkinson’s Disease Using RapidFire Mass Spectrometry

High-Throughput Screening Using Mass Spectrometry within Drug Discovery

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