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

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.

SUBMITTER: Steger M

PROVIDER: S-EPMC8438043 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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

Steger Martin M Demichev Vadim V Backman Mattias M Ohmayer Uli U Ihmor Phillip P Müller Stefan S Ralser Markus M Daub Henrik H

Nature communications 20210913 1

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 ...[more]

PMID: 34518535

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Project description:Ubiquitination has crucial roles in many cellular processes, and dysregulation of ubiquitin machinery enzymes can result in various forms of pathogenesis. Cells only have a limited set of ubiquitin-conjugating (E2) enzymes to support the ubiquitination of many cellular targets. As individual E2 enzymes have many different substrates and interactions between E2 enzymes and their substrates can be transient, it is challenging to define all in vivo substrates of an individual E2 and the cellular processes it affects. Particularly challenging in this respect is UBE2D3, an E2 enzyme with promiscuous activity in vitro but less defined roles in vivo. Here, we set out to identify in vivo targets of UBE2D3 by using stable isotope labeling by amino acids in cell culture-based and label-free quantitative ubiquitin diGly proteomics to study global proteome and ubiquitinome changes associated with UBE2D3 depletion. UBE2D3 depletion changed the global proteome, with the levels of proteins from metabolic pathways, in particular retinol metabolism, being the most affected. However, the impact of UBE2D3 depletion on the ubiquitinome was much more prominent. Interestingly, molecular pathways related to mRNA translation were the most affected. Indeed, we find that ubiquitination of the ribosomal proteins RPS10 and RPS20, critical for ribosome-associated protein quality control, is dependent on UBE2D3. We show by Targets of Ubiquitin Ligases Identified by Proteomics 2 methodology that RPS10 and RPS20 are direct targets of UBE2D3 and demonstrate that the catalytic activity of UBE2D3 is required to ubiquitinate RPS10 in vivo. In addition, our data suggest that UBE2D3 acts at multiple levels in autophagic protein quality control. Collectively, our findings show that depletion of an E2 enzyme in combination with quantitative diGly-based ubiquitinome profiling is a powerful tool to identify new in vivo E2 substrates, as we have done here for UBE2D3. Our work provides an important resource for further studies on the in vivo functions of UBE2D3.

Project description:BackgroundVaccine development against malaria and other complex diseases remains a challenge for the scientific community. The recent elucidation of the genome, proteome and transcriptome of many of these complex pathogens provides the basis for rational vaccine design by identifying, on a proteome-wide scale, novel target antigens that are recognized by T cells and antibodies from exposed individuals. However, there is currently no algorithm to effectively identify important target antigens from genome sequence data; this is especially challenging for T cell targets. Furthermore, for some of these pathogens, such as Plasmodium, protein expression using conventional platforms has been problematic but cell-free in vitro transcription translation (IVTT) strategies have recently proved successful. Herein, we report a novel approach for proteome-wide scale identification of the antigenic targets of T cell responses using IVTT products.Principal findingsWe conducted a series of in vitro and in vivo experiments using IVTT proteins either unpurified, absorbed to carboxylated polybeads, or affinity purified through nickel resin or magnetic beads. In vitro studies in humans using CMV, EBV, and Influenza A virus proteins showed antigen-specific cytokine production in ELIspot and Cytometric Bead Array assays with cells stimulated with purified or unpurified IVTT antigens. In vitro and in vivo studies in mice immunized with the Plasmodium yoelii circumsporozoite DNA vaccine with or without IVTT protein boost showed antigen-specific cytokine production using purified IVTT antigens only. Overall, the nickel resin method of IVTT antigen purification proved optimal in both human and murine systems.ConclusionsThis work provides proof of concept for the potential of high-throughput approaches to identify T cell targets of complex parasitic, viral or bacterial pathogens from genomic sequence data, for rational vaccine development against emerging and re-emerging diseases that pose a threat to public health.

Dataset Information

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

Publications

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

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