Abstract
Protein ubiquitylation is an essential mechanism regulating almost all cellular functions in eukaryotes. The understanding of the role of distinct ubiquitin chains in different cellular processes is essential to identify biomarkers for disease diagnosis and prognosis but also to open new therapeutic possibilities. The high complexity of ubiquitin chains complicates this analysis, and multiple strategies have been developed over the last decades. Here, we report a protocol for the isolation and identification of K48 and K63 ubiquitin chains using chain-specific nanobodies associated to mass spectrometry. Different steps were optimized to increase the purification yield and reduce the binding on nonspecific proteins. The resulting protocol allows the enrichment of ubiquitin chain-specific targets from mammalian cells.
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Acknowledgments
We thank all members of the UbiCARE lab, Clémence Coutelle-Rebut for the proofreading of this manuscript. MGS is a fellow from the UbiCODE project funded by the EU’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement (No 765445) and the Association contre le Cancer (ARC). We acknowledge the COST Action CA20113 “PROTEOCURE” supported by COST (European Cooperation in Science and Technology). MSR was also funded by CONACyT-SRE (Mexico) grant 0280365 and the REPERE and prématuration (Ubipièges) programs of Occitanie.
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Gonzalez-Santamarta, M., Ceccato, L., Carvalho, A.S., Rain, JC., Matthiesen, R., Rodriguez, M.S. (2023). Isolation and Mass Spectrometry Identification of K48 and K63 Ubiquitin Proteome Using Chain-Specific Nanobodies. In: Rodriguez, M.S., Barrio, R. (eds) The Ubiquitin Code. Methods in Molecular Biology, vol 2602. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2859-1_9
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DOI: https://doi.org/10.1007/978-1-0716-2859-1_9
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Online ISBN: 978-1-0716-2859-1
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