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Application note: High-throughput Screening of Deubiquitylase enzyme (DUB) activity/specificity and inhibitor screening by MALDI-TOF mass spectrometry
1 September 2015 • Author(s): Bruker Daltonik
In biopharmaceutical laboratories, there is a growing need for high-throughput, yet sensitive methodologies to characterize deubiquitylase enzymes (DUBs) and DUB inhibitors.
Current high-throughput screening (HTS) approaches make use of fluorogenic substrates or chemical probes, which do not represent real physiological substrates, thus leading to potential false-positives or false-negatives. We have developed a sensitive and fast assay to quantify in vitro DUB enzyme activity using matrixassisted laser desorption/ionization time-of-flight (MALDITOF) mass spectrometry, called MALDI-TOF DUB assay. Unlike other current assays, this method uses unmodified substrates, such as diubiquitin topoisomers.
By using of MALDI-TOF and a 15N-Ubiquitin internal standard, we are able to isotopically resolve the monoubiquitin formed by DUB at the intact protein level, and thus robustly quantify the production of this monoubiquitin. We demonstrate high linearity over almost three orders of magnitude with very high intraday/interday reproducibility (CV<10%). The MALDITOF DUB assay now enables an extensive characterization of DUB activity and specificity as well as the assessment of the potency and specificity of DUB inhibitors in a highthroughput manner.
ABB Analytical Measurement ACD/Labs ADInstruments Ltd Advanced Analytical Technologies GmbH Analytik Jena AG Astell Scientific Ltd B&W Tek Bachem AG Bibby Scientific Limited Bio-Rad Laboratories BioNavis Ltd Biopharma Group Black Swan Analysis Limited CAPSUGEL NV Charles Ischi AG | Kraemer Elektronik Cherwell Laboratories CI Precision Cobalt Light Systems Coulter Partners CPC Biotech srl Dassault Systèmes BIOVIA DiscoverX Edinburgh Instruments Enterprise System Partners (ESP) Eurofins BioPharma Product Testing EUROGENTEC F.P.S. Food and Pharma Systems Srl GE Analytical Instruments IDBS JEOL Europe L.B. Bohle Maschinen + Verfahren GmbH Lab M Ltd. LabWare Linkam Scientific Instruments Limited MA Business Metrohm Molins Technologies Multicore Dynamics Ltd Nanosurf New England Biolabs, Inc. Ocean Optics Panasonic Biomedical Sales Europe B.V. PerkinElmer Inc ReAgent Russell Finex Limited Source BioScience Takara Clontech Tornado Spectral Systems Tuttnauer Viavi Solutions, Inc Watson-Marlow Fluid Technology Group Wickham Laboratories Limited Xylem Analytics YMC Europe GmbH Yusen Logistics