Application note: High-throughput Screening of Deubiquitylase enzyme (DUB) activity/specificity and inhibitor screening by MALDI-TOF mass spectrometry
Posted: 1 September 2015 | Bruker
In biopharmaceutical laboratories, there is a growing need for high-throughput, yet sensitive methodologies to characterize deubiquitylase enzymes (DUBs) and DUB inhibitors…
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.