Specific and Unbiased Detection of Polyubiquitination via a Sensitive Non-Antibody Approach.,Analytical Chemistry

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Specific and Unbiased Detection of Polyubiquitination via a Sensitive Non-Antibody Approach.
Analytical Chemistry ( IF 6.7 ) Pub Date : 2019-12-10 , DOI: 10.1021/acs.analchem.9b04092
Weidi Xiao ₁ , Zijuan Liu _{1,

2} , Weijia Luo ₁ , Yuan Gao ₁ , Lei Chang ₁ , Yanchang Li ₁ , Ping Xu _{1,

2,

3,

4}

Affiliation

Polyubiquitination encompasses complex topologies through various linkage types to deliver diverse cellular signals, which has been recognized as a sophisticated ubiquitin code. Accurate comparison of polyubiquitination signals is critical for revealing the dynamic cellular ubiquitination-regulated events. Western blotting (WB) is the most widely used biochemical method to quantify proteins and posttranslational modifications under diverse physiological conditions. The accuracy and sensitivity of the WB mainly depend on the quality and specificity of the antibody. In this study, we found that the antiubiquitin antibodies exhibited different affinities to the eight linkage types of ubiquitin chains, with the highest sensitivity for the K63-linked chain, lower efficiency for M1 and K48, and very low affinity for the other types of chains. Herein, we introduced the tandem hybrid ubiquitin-binding domain (ThUBD)-based far-Western blotting (TUF-WB) to visualize the signal of synthetic ubiquitin chains or ubiquitinated conjugates on a solid membrane by utilizing the unbiased affinity of ThUBD to all types of ubiquitin linkages. As compared to antiubiquitin antibody detection, TUF-WB can accurately quantify the signal intensity to the mass amounts of all eight ubiquitin chains. Meanwhile, the sensitivity of this method in detecting complex ubiquitinated samples was 4-5-fold higher than those of antibodies. Consequently, TUF-WB allows accurate quantification of polyubiquitination signal on the membrane with great sensitivity and wider dynamic range.

中文翻译：

通过灵敏的非抗体方法对多泛素化的特异性和无偏性检测。

多泛素化通过各种链接类型涵盖复杂的拓扑结构，以传递各种细胞信号，这已被公认为是复杂的泛素代码。准确比较多泛素化信号对于揭示动态细胞泛素化调节事件至关重要。Western blotting（WB）是最广泛使用的生化方法，用于在各种生理条件下定量蛋白质和翻译后修饰。WB的准确性和敏感性主要取决于抗体的质量和特异性。在这项研究中，我们发现抗泛素抗体对八种泛素链的连接类型表现出不同的亲和力，对K63连接的链的敏感性最高，对M1和K48的效率较低，对其他类型的链的亲和力很低。在此处，我们介绍了基于串联杂交的遍在蛋白结合域（ThUBD）的远西印迹（TUF-WB），通过利用ThUBD对所有类型的遍在蛋白的无偏亲和力来可视化合成的遍在蛋白链或遍在蛋白在固体膜上的信号联系。与抗泛素抗体检测相比，TUF-WB可以精确定量所有八个泛素链质量的信号强度。同时，该方法检测复杂的泛素化样品的灵敏度比抗体高4-5倍。因此，TUF-WB可以以高灵敏度和更宽的动态范围准确定量膜上的多泛素化信号。

更新日期：2019-12-11

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