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Lookup NU author(s): Dr Tiaan Heunis, Dr Frederic Lamoliatte, Dr Jose Luis Marin-RubioORCiD, Dr Abeer Dannoura, Professor Matthias TrostORCiD
This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).
© 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.
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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