TY  - JOUR
A1  - Pinto-Fernández, Adán
A1  - Davis, Simon
A1  - Schofield, Abigail B.
A1  - Scott, Hannah C.
A1  - Zhang, Ping
A1  - Salah, Eidarus
A1  - Mathea, Sebastian
A1  - Charles, Philip D.
A1  - Damianou, Andreas
A1  - Bond, Gareth
A1  - Fischer, Roman
A1  - Kessler, Benedikt
T1  - Comprehensive landscape of active deubiquitinating enzymes profiled by advanced chemoproteomics
T2  - Frontiers in Chemistry
N2  - Enzymes that bind and process ubiquitin, a small 76-amino-acid protein, have been recognized as pharmacological targets in oncology, immunological disorders, and neurodegeneration. Mass spectrometry technology has now reached the capacity to cover the proteome with enough depth to interrogate entire biochemical pathways including those that contain DUBs and E3 ligase substrates. We have recently characterized the breast cancer cell (MCF7) deep proteome by detecting and quantifying ~10,000 proteins, and within this data set, we can detect endogenous expression of 65 deubiquitylating enzymes (DUBs), whereas matching transcriptomics detected 78 DUB mRNAs. Since enzyme activity provides another meaningful layer of information in addition to the expression levels, we have combined advanced mass spectrometry technology, pre-fractionation, and more potent/selective ubiquitin active-site probes with propargylic-based electrophiles to profile 74 DUBs including distinguishable isoforms for 5 DUBs in MCF7 crude extract material. Competition experiments with cysteine alkylating agents and pan-DUB inhibitors combined with probe labeling revealed the proportion of active cellular DUBs directly engaged with probes by label-free quantitative (LFQ) mass spectrometry. This demonstrated that USP13, 39, and 40 are non-reactive to probe, indicating restricted enzymatic activity under these cellular conditions. Our extended chemoproteomics workflow increases depth of covering the active DUBome, including isoform-specific resolution, and provides the framework for more comprehensive cell-based small-molecule DUB selectivity profiling.
KW  - deubiquitylating enzymes
KW  - mass spectrometry
KW  - proteomics
KW  - chemical biology
KW  - ubiquitin specific proteases
KW  - isoforms
Y1  - 2019
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/51101
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-511016
SN  - 2296-2646
N1  - Copyright © 2019 Pinto-Fernández, Davis, Schofield, Scott, Zhang, Salah, Mathea, Charles, Damianou, Bond, Fischer and Kessler. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
VL  - 7
IS  - Art. 592
SP  - 1
EP  - 14
PB  - Frontiers Media
CY  - Lausanne
ER  -