Elu Nagore, Lectez Benoit, Ramirez Juanma, Osinalde Nerea, Mayor Ugo
Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), Leioa, Spain.
Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of the Basque Country (UPV/EHU), Vitoria-Gasteiz, Spain.
Methods Mol Biol. 2020;2051:265-276. doi: 10.1007/978-1-4939-9744-2_11.
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.
泛素（Ub）和类泛素（UbL）修饰剂的调节作用可为其底物蛋白赋予无数功能，比如诱导蛋白质-蛋白质相互作用、促使膜蛋白内化，或者通过蛋白酶体使其降解。调控这些不同结果的潜在密码似乎基于所形成的泛素链的拓扑结构。由Ub和UbL介导的特定调节作用的实验表征并非易事。Ub和UbL修饰蛋白的亚化学计量水平极大地限制了它们在更为丰富的蛋白质背景中的分析检测。因此，在进行任何下游检测分析之前，必须先富集修饰的蛋白质或肽段。为此，我们最近开发了一种基于绿色荧光蛋白（GFP）标签的分离策略。在此，我们阐述了将GFP标签分离策略与质谱（MS）相结合，以鉴定GFP标签蛋白内的Ub和UbL修饰残基，以及揭示所形成的Ub和UbL链类型的实用性。
