Abstract
Regulation by ubiquitin (Ub) and ubiquitin-like (UbL) modifiers can confer their substrate proteins a myriad of assignments, such as inducing protein–protein interactions, the internalization of membrane proteins, or their degradation via the proteasome. The underlying code regulating those diverse endpoints appears to be based on the topology of the ubiquitin chains formed.
Experimental characterization of the specific regulation mediated by Ub and UbLs is not trivial. The substoichiometric levels of Ub- and UbL-modified proteins greatly limit their analytical detection in a background of more abundant proteins. Therefore, modified proteins or peptides must be enriched prior to any downstream detection analysis. For that purpose, we recently developed a GFP-tag based isolation strategy. Here we illustrate the usefulness of combining GFP-tag isolation strategy with mass spectrometry (MS) to identify Ub- and UbL-modified residues within the GFP-tagged protein, as well as to uncover the types of Ub and UbL chains formed.
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Acknowledgments
We would like to acknowledge Jabi Beaskoetxea, Kerman Aloria, and Jesus Mari Arizmendi for all their advice and support. The authors are grateful of the technical support provided by UPV/EHU SGIker (ERDF and ESF). This work was supported by Spanish MINECO [grant SAF2016-76898-P].
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Elu, N., Lectez, B., Ramirez, J., Osinalde, N., Mayor, U. (2020). Mass Spectrometry-Based Characterization of Ub- and UbL-Modified Proteins. In: Matthiesen, R. (eds) Mass Spectrometry Data Analysis in Proteomics. Methods in Molecular Biology, vol 2051. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9744-2_11
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DOI: https://doi.org/10.1007/978-1-4939-9744-2_11
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