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Technical report: Targeted proteomic analysis reveals enrichment of atypical ubiquitin chains in contractile murine tissues

Lookup NU author(s): Dr Tiaan Heunis, Dr Frederic Lamoliatte, Dr Jose Marin Rubio, Dr Abeer Dannoura, Professor Matthias Trost

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


Abstract

© 2020 The AuthorsUbiquitylation is an elaborate post-translational modification involved in all biological processes. Its pleotropic effect is driven by the ability to form complex polyubiquitin chain architectures that can influence biological functions. In this study, we optimised sample preparation and chromatographic separation of Ubiquitin peptides for Absolute Quantification by Parallel Reaction Monitoring (Ub-AQUA-PRM). Using this refined Ub-AQUA-PRM assay, we were able to quantify all ubiquitin chain types in 10-min LC-MS/MS runs. We used this method to determine the ubiquitin chain-linkage composition in murine bone marrow-derived macrophages and different mouse tissues. We could show tissue-specific differences in ubiquitin levels in murine tissues, with polyubiquitin chain types contributing a small proportion to the total pool of ubiquitin. Interestingly, we observed enrichment of atypical (K33) ubiquitin chains in heart and muscle. Our approach enabled high-throughput screening of ubiquitin chain-linkage composition in different murine tissues and highlighted a possible role for atypical ubiquitylation in contractile tissues. Significance: Large knowledge gaps exist in our understanding of ubiquitin chain-linkage composition in mammalian tissues. Defining this in vivo ubiquitin chain-linkage landscape could reveal the functional importance of different ubiquitin chain types in tissues. In this study, we refined the previously described Ub-AQUA-PRM assay to enable quantification of all ubiquitin chain types in a high-throughput manner. Using this assay, we provided new data on the ubiquitin chain-linkage composition in primary murine macrophages and tissues, and revealed an enrichment of atypical ubiquitin chains in contractile tissues. Our approach should thus enable rapid, high-throughput screening of ubiquitin chain-linkage composition in different sample types, as demonstrated in murine primary cells and tissues.


Publication metadata

Author(s): Heunis T, Lamoliatte F, Marin-Rubio JL, Dannoura A, Trost M

Publication type: Article

Publication status: Published

Journal: Journal of Proteomics

Year: 2020

Volume: 229

Print publication date: 30/10/2020

Online publication date: 06/09/2020

Acceptance date: 24/08/2020

Date deposited: 04/12/2020

ISSN (print): 1874-3919

ISSN (electronic): 1876-7737

Publisher: Elsevier B.V.

URL: https://doi.org/10.1016/j.jprot.2020.103963

DOI: 10.1016/j.jprot.2020.103963

PubMed id: 32898700


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