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

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 netw...

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Bibliographic Details
Published in:Nature communications 2021-09, Vol.12 (1), p.5399-5399, Article 5399
Main Authors: Steger, Martin, Demichev, Vadim, Backman, Mattias, Ohmayer, Uli, Ihmor, Phillip, Müller, Stefan, Ralser, Markus, Daub, Henrik
Format: Article
Language:English
Subjects:
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Data processing
Deep learning
Drug delivery
Drug development
Humanities and Social Sciences
Information processing
Mass spectrometry
Mass spectroscopy
multidisciplinary
Neural networks
Peptides
Proteins
Proteomes
Sample preparation
Science
Science (multidisciplinary)
Scientific imaging
Spectroscopy
Temporal resolution
Ubiquitin
Ubiquitination
Workflow
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Summary: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. Combining improved sample preparation, data-independent acquisition mass spectrometry and deep learning, the authors develop a workflow for more robust and precise quantitative ubiquitinome profiling. They use this method to characterize targets of the deubiquitinase USP7 and effects of USP7 inhibitors.
ISSN:2041-1723
2041-1723
DOI:10.1038/s41467-021-25454-1