Abstract
Ubiquitylation (or ubiquitination) is the reversible conjugation of a 76-amino-acid polypeptide (ubiquitin) to a target protein to modulate various biological processes. Deubiquitylating enzymes (DUBs) are a class of enzymes that specifically remove ubiquitin from a substrate. In recent years DUBs have garnered significant attention as a new class of targets in multiple therapeutic areas. The recent development of high-throughput Matrix-Assisted Laser Desorption/Ionization-Time of Flight mass spectrometry (MALDI-TOF MS) has provided new tools to perform drug discovery screening. Here we present a facile and high-throughput step-by-step protocol of the MALDI-TOF MS-based DUB assay for screening the activity of DUBs in vitro. In a MALDI-TOF DUB assay, we quantitate the amount of mono-ubiquitin generated by the in vitro cleavage of ubiquitin chains. The presented protocol takes advantage of nanoliter dispensing robotics and automated MALDI-TOF MS analysis to screen large and diverse compound libraries.
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