Specific and Unbiased Detection of Polyubiquitination via a Sensitive Non-Antibody Approach.
Specific and Unbiased Detection of Polyubiquitination via a Sensitive Non-Antibody Approach.
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通过灵敏的非抗体方法特异性且公正地检测多泛素化。
DOI：
10.1021/acs.analchem.9b04092
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发表时间：
2019
影响因子：
7.4
通讯作者：
Xu Ping
中科院分区：
文献类型：
--
作者：
Xiao Weidi;Liu Zijuan;Luo Weijia;Gao Yuan;Chang Lei;Li Yanchang;Xu Ping
多泛素化包含通过各种连接类型的复杂拓扑，以传递不同的细胞信号，这已被认为是复杂的泛素代码。准确比较多聚泛素化信号对于揭示动态细胞泛素化调控事件至关重要。蛋白质印迹 (WB) 是最广泛使用的生化方法，用于在不同的生理条件下定量蛋白质和翻译后修饰。 WB的准确性和灵敏度主要取决于抗体的质量和特异性。在本研究中，我们发现抗泛素抗体对八种连接类型的泛素链表现出不同的亲和力，对K63连接的链具有最高的敏感性，对M1和K48的效率较低，对其他类型的链的亲和力非常低。在此，我们引入了基于串联杂交泛素结合结构域 (ThUBD) 的远蛋白印迹 (TUF-WB)，利用 ThUBD 对所有类型泛素连接的无偏亲和力，可视化固体膜上合成泛素链或泛素化缀合物的信号。与抗泛素抗体检测相比，TUF-WB 可以准确量化所有八条泛素链质量的信号强度。同时，该方法检测复杂泛素化样品的灵敏度比抗体高4-5倍。因此，TUF-WB 能够以极高的灵敏度和更宽的动态范围准确定量膜上的多聚泛素化信号。
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.