title: Demonstrating enhanced throughput of RapidFire mass spectrometry through multiplexing using the JmjD2d demethylase as a model system.
abstract: Using mass spectrometry to detect enzymatic activity offers several advantages over fluorescence-based methods. Automation of sample handling and analysis using platforms such as the RapidFire (Agilent Technologies, Lexington, MA) has made these assays amenable to medium-throughput screening (of the order of 100,000 wells). However, true high-throughput screens (HTS) of large compound collections (>1 million) are still considered too time-consuming to be feasible. Here we propose a simple multiplexing strategy that can be used to increase the throughput of RapidFire, making it viable for HTS. The method relies on the ability to analyze pooled samples from several reactions simultaneously and to deconvolute their origin using "mass-tagged" substrates. Using the JmjD2d H3K9me3 demethylase as a model system, we demonstrate the practicality of this method to achieve a 4-fold increase in throughput. This was achieved without any loss of assay quality. This multiplex strategy could easily be scaled to give even greater reductions in analysis time.
authors.affiliation: 1Department of Screening and Compound Profiling, GlaxoSmithKline, Stevenage, UK.
authors.forename: Melanie
authors.initials: M
authors.lastname: Leveridge
authors.forename: Rachel
authors.initials: R
authors.lastname: Buxton
authors.forename: Argyrides
authors.initials: A
authors.lastname: Argyrou
authors.forename: Peter
authors.initials: P
authors.lastname: Francis
authors.forename: Bill
authors.initials: B
authors.lastname: Leavens
authors.forename: Andy
authors.initials: A
authors.lastname: West
authors.forename: Mike
authors.initials: M
authors.lastname: Rees
authors.forename: Philip
authors.initials: P
authors.lastname: Hardwicke
authors.forename: Angela
authors.initials: A
authors.lastname: Bridges
authors.forename: Steven
authors.initials: S
authors.lastname: Ratcliffe
authors.forename: Chun-wa
authors.initials: CW
authors.lastname: Chung
mesh_terms: D057890:Epigenomics; D057166:High-Throughput Screening Assays; D006801:Humans; D056484:Jumonji Domain-Containing Histone Demethylases / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D056484:Jumonji Domain-Containing Histone Demethylases; C521694:KDM4D protein, human
keywords: Jumonji enzymes; RapidFire; epigenetics; high-throughput screen; histone demethylase; mass spectrometry; multiplexing