Using Activity-Based Proteomics for the Quantification of Deubiquitinases in Animal Tissue.

Autor:	Nanduri B; Department of Comparative Biomedical Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, USA., Shack LA; Department of Comparative Biomedical Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, USA., Santelices J; Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, USA., Edelmann MJ; Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, USA. medelmann@ufl.edu.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2023; Vol. 2591, pp. 45-57.
DOI:	10.1007/978-1-0716-2803-4_4
Abstrakt:	Ubiquitination is a post-translational modification, that regulates essential cellular functions, and the enzymes that control the removal of this modification, deubiquitinases (DUBs), have been well described for the model organisms. However, the information about DUBs is still largely lacking for the non-model organisms, such as agriculturally relevant animals. To understand the expression of these enzymes in animal tissues, we have used chemical proteomics which can be used to identify biologically active DUBs present in tissues based on their reactivity with the activity-based probes (ABPs). Here we describe a sample preparation protocol for ABP-based purification of DUBs from animal tissue using two approaches to homogenize and lyse the animal tissue compatible with ABP labeling of DUBs, including an ultrasonication-based tissue processing method and bead-beating method. Both of these methods retain the enzymatic activity of DUBs. In addition, we describe a protocol for ABP labeling of DUBs in tissue lysates and the immunoprecipitation of the probe-reactive DUBs that can be used along with mass spectrometric identification of proteins and the detection of these DUBs by Western blotting. (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze:	MEDLINE
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