Systematic approach for validating the ubiquitinated proteome
Systematic approach for validating the ubiquitinated proteome
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DOI：
10.1021/ac702516a
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发表时间：
2008-06-01
影响因子：
7.4
通讯作者：
Peng, Junmin
中科院分区：
文献类型：
--
作者：
Seyfried, Nicholas T.;Xu, Ping;Peng, Junmin
蛋白质泛素化在所有真核生物中起着重要的调节作用。泛素化蛋白的大规模分析通常是通过结合亲和纯化策略和质谱法进行的。然而，目前还没有可靠的方法来系统地区分泛素化和未修饰的成分。在这里，我们报告了一种简单的策略，通过重建凝胶电泳和质谱分析蛋白质的虚拟Western blots，大规模验证泛素化。由于蛋白质泛素化，特别是多泛素化会引起分子量的剧烈变化，因此我们使用实验分子量与预期分子量之间的差异来确定泛素化的状态。利用高斯曲线拟合的方法，根据凝胶中光谱计数的值和分布计算了酵母泛素偶联物的实验分子量。还分析了酵母细胞裂解液中未修饰的蛋白质作为对照，以评估该方法的准确性。结合泛素质量和/或实验变异的多个阈值评估了敏感性和特异性。最终，根据严格的过滤标准，只有接近30%的候选泛素偶联物被接受，尽管它们是在变性条件下纯化的。这些被接受的偶联物估计有8%的错误发现率，主要由大于100 kDa的蛋白质组成。与另一种验证方法（即鉴定泛素化赖氨酸位点）相比，在虚拟Western blots上，接近95%具有确定修饰位点的蛋白质显示出令人信服的分子量增加。第二个独立分析表明，该方法可以通过切除少于10个凝胶带来简化。因此，这种策略为泛素化蛋白的解释建立了必要的标准。
Protein ubiquitination plays an essential regulatory role within all eukaryotes. Large-scale analyses of ubiquitinated proteins are usually performed by combining affinity purification strategies with mass spectrometry. However, there is no reliable method to systematically differentiate ubiquitinated species from copurified unmodified components. Here we report a simple strategy for the large-scale validation of ubiquitination by reconstructing virtual Western blots for proteins analyzed by gel electrophoresis and mass spectrometry. Because protein ubiquitination, especially polyubiquitination, causes a dramatic shift of molecular weight, the difference between experimental and expected molecular weight was used to confirm the status of ubiquitination. Experimental molecular weight of putative yeast ubiquitin-conjugates was computed from the value and distribution of spectral counts in the gel using a Gaussian curve fitting approach. Unmodified proteins in yeast cell lysate were also analyzed as a control to assess the accuracy of the method. Multiple thresholds that incorporated the mass of ubiquitin and/or experimental variations were evaluated with respect to sensitivity and specificity. Ultimately, only similar to 30% of the candidate ubiquitin-conjugates were accepted based on the stringent filtering criteria, although they were purified under denaturing conditions. These accepted conjugates had an estimated false discovery rate of similar to 8% and primarily consisted of proteins larger than 100 kDa. Compared with another validation method (i.e., identification of ubiquitinated lysine sites), similar to 95% of the proteins with defined modification sites showed a convincing increase in molecular weight on the virtual Western blots. A second independent analysis indicated that the method can be simplified by excising fewer than ten gel bands. Therefore, this strategy establishes criteria necessary for the interpretation of ubiquitinated proteins.