CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, Nijmegen, the Netherlands.
Nat Protoc. 2018 Mar;13(3):530-550. doi: 10.1038/nprot.2017.147. Epub 2018 Feb 15.
Ubiquitin-binding proteins play an important role in eukaryotes by translating differently linked polyubiquitin chains into proper cellular responses. Current knowledge about ubiquitin-binding proteins and ubiquitin linkage-selective interactions is mostly based on case-by-case studies. We have recently reported a method called ubiquitin interactor affinity enrichment-mass spectrometry (UbIA-MS), which enables comprehensive identification of ubiquitin interactors for all ubiquitin linkages from crude cell lysates. One major strength of UbIA-MS is the fact that ubiquitin interactors are enriched from crude cell lysates, in which proteins are present at endogenous levels, contain biologically relevant post-translational modifications (PTMs) and are assembled in native protein complexes. In addition, UbIA-MS uses chemically synthesized nonhydrolyzable diubiquitin, which mimics native diubiquitin and is inert to cleavage by endogenous deubiquitinases (DUBs). Here, we present a detailed protocol for UbIA-MS that proceeds in five stages: (i) chemical synthesis of ubiquitin precursors and click chemistry for the generation of biotinylated nonhydrolyzable diubiquitin baits, (ii) in vitro affinity purification of ubiquitin interactors, (iii) on-bead interactor digestion, (iv) liquid chromatography (LC)-MS/MS analysis and (v) data analysis to identify differentially enriched proteins. The computational analysis tools are freely available as an open-source R software package, including a graphical interface. Typically, UbIA-MS allows the identification of dozens to hundreds of ubiquitin interactors from any type of cell lysate, and can be used to study cell type or stimulus-dependent ubiquitin interactions. The nonhydrolyzable diubiquitin synthesis can be completed in 3 weeks, followed by ubiquitin interactor enrichment and identification, which can be completed within another 2 weeks.
泛素结合蛋白通过将不同连接的多泛素链翻译为适当的细胞反应，在真核生物中发挥重要作用。目前关于泛素结合蛋白和泛素连接选择性相互作用的知识主要基于个案研究。我们最近报道了一种称为泛素相互作用蛋白亲和富集-质谱（UbIA-MS）的方法，该方法能够从粗细胞裂解物中全面鉴定所有泛素连接的泛素相互作用蛋白。UbIA-MS 的一个主要优势是，泛素相互作用蛋白是从粗细胞裂解物中富集的，其中蛋白质以内源性水平存在，含有生物学相关的翻译后修饰（PTMs），并以天然蛋白复合物的形式组装。此外，UbIA-MS 使用化学合成的不可水解的二泛素，该二泛素模拟天然二泛素，并且对内源性去泛素酶（DUBs）的切割无反应活性。在这里，我们提供了 UbIA-MS 的详细方案，该方案分为五个阶段：（i）泛素前体的化学合成和点击化学用于生成生物素化的不可水解的二泛素诱饵，（ii）体外泛素相互作用蛋白亲和纯化，（iii）珠上相互作用蛋白消化，（iv）液相色谱（LC）-MS/MS 分析和（v）数据分析以鉴定差异富集的蛋白质。计算分析工具作为开源 R 软件包免费提供，包括一个图形界面。通常，UbIA-MS 可以从任何类型的细胞裂解物中鉴定数十到数百种泛素相互作用蛋白，并且可用于研究细胞类型或刺激依赖性的泛素相互作用。不可水解的二泛素合成可以在 3 周内完成，随后是泛素相互作用蛋白的富集和鉴定，这可以在另外 2 周内完成。
