High-throughput matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry–based deubiquitylating enzyme assay for drug discovery

Abstract

Deubiquitylating enzymes (DUBs) play a vital role in the ubiquitin pathway by editing or removing ubiquitin from their substrate. As breakthroughs within the ubiquitin field continue to highlight the potential of deubiquitylating enzymes as drug targets, there is increasing demand for versatile high-throughput (HT) tools for the identification of potent and selective DUB modulators. Here we present the HT adaptation of the previously published MALDI-TOF-based DUB assay method. In a MALDI-TOF DUB assay, we quantitate the amount of mono-ubiquitin generated by the in vitro cleavage of ubiquitin chains by DUBs. The method has been specifically developed for use with nanoliter-dispensing robotics to meet drug discovery requirements for the screening of large and diverse compound libraries. Contrary to the most common DUB screening technologies currently available, the MALDI-TOF DUB assay combines the use of physiological substrates with the sensitivity and reliability of the mass spectrometry-based readout.

Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies

“…Importantly the bioactivity and safety of these compounds has been tested in clinical trials allowing rapid repurposing of any hit compounds into therapies for the treatment of COVID-19. Compared to other fluorescent-based approaches, the MALDI-TOF-based assay has the advantage of using K48 ubiquitin trimer, a physiological PLpro substrate, for high-throughput screening (HTS) [40]. Further, the final products generated upon cleavage of triubiquitin are diubiquitin and monoubiquitin, which makes it compatible for analysis using the MALDI DUB assay.…”

“…Enzymatic reactions and MALDI-TOF High-Throughput Screening (HTS) were set up in assay as previously described [40]. Briefly, FDA approved compound library was delivered into 384 well plated plates using non-contact acoustic delivering.…”

A MALDI-TOF assay identifies nilotinib as an inhibitor of inflammation in acute myeloid leukaemia

“…In recent years, MALDI-TOF MS has become an exciting new tool for drug discovery for in vitro assays. 35–37 Here, we applied a screen in whole cells, allowing the identification of biomolecules that provide a specific fingerprint for the phenotype of the analyzed cells. 38 Two features at m/z 4632 and 4964 were significantly altered upon inflammatory stimuli.…”

“…53,54 As the technology allows very fast screening, it has already found traction in the HTS field with its application to label-free screening of in vitro assays. 13,15,16,55–59…”

Ubiquitination and Deubiquitination in Oral Disease

“…Deubiquitinating enzymes (DUBs) are proteases that reverse protein ubiquitination, a process that is important in normal homeostasis, or trim Ub chains of diverse linkages [ 10 , 11 , 12 ]. The variety of cellular processes initiated and regulated by ubiquitin has been explained in part by the structural diversity of differently linked ubiquitin chains [ 13 ].…”