Time-resolved in vivo ubiquitinome profiling by DIA-MS reveals USP7 targets on a proteome-wide scale

Martin Steger (), Vadim Demichev, Mattias Backman, Uli Ohmayer, Phillip Ihmor, Stefan Müller, Markus Ralser and Henrik Daub
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Martin Steger: Evotec München GmbH, Am Klopferspitz 19a
Vadim Demichev: Charité – Universitätsmedizin Berlin, Department of Biochemistry
Mattias Backman: Evotec München GmbH, Am Klopferspitz 19a
Uli Ohmayer: Evotec München GmbH, Am Klopferspitz 19a
Phillip Ihmor: Evotec München GmbH, Am Klopferspitz 19a
Stefan Müller: Evotec München GmbH, Am Klopferspitz 19a
Markus Ralser: Charité – Universitätsmedizin Berlin, Department of Biochemistry
Henrik Daub: Evotec München GmbH, Am Klopferspitz 19a

Nature Communications, 2021, vol. 12, issue 1, 1-13

Abstract: Abstract Mass spectrometry (MS)-based ubiquitinomics provides system-level understanding of ubiquitin signaling. Here we present a scalable workflow for deep and precise in vivo ubiquitinome profiling, coupling an improved sample preparation protocol with data-independent acquisition (DIA)-MS and neural network-based data processing specifically optimized for ubiquitinomics. Compared to data-dependent acquisition (DDA), our method more than triples identification numbers to 70,000 ubiquitinated peptides in single MS runs, while significantly improving robustness and quantification precision. Upon inhibition of the oncology target USP7, we simultaneously record ubiquitination and consequent changes in abundance of more than 8,000 proteins at high temporal resolution. While ubiquitination of hundreds of proteins increases within minutes of USP7 inhibition, we find that only a small fraction of those are ever degraded, thereby dissecting the scope of USP7 action. Our method enables rapid mode-of-action profiling of candidate drugs targeting DUBs or ubiquitin ligases at high precision and throughput.

Date: 2021
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DOI: 10.1038/s41467-021-25454-1

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