Two-dimensional electrophoresis-based characterization of post-translational modifications of mammalian 20S proteasome complexes.
Two-dimensional electrophoresis-based characterization of post-translational modifications of mammalian 20S proteasome complexes.
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DOI：
10.1002/pmic.200800387
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发表时间：
2008-12
期刊：
影响因子：
3.4
通讯作者：
Ping, Peipei
中科院分区：
文献类型：
--
作者：
Zong, Chenggong;Young, Glen W.;Wang, Yueju;Lu, Haojie;Deng, Ning;Drews, Oliver;Ping, Peipei
关键词：
翻译后修饰（PTMs）是20S蛋白酶体功能的关键调控机制。先前已经观察到20S ptm的改变，改变了蛋白质降解模式和改变的细胞表型。尽管经过数十年的研究，我们对20S复合物的各种ptm及其生物学意义的了解仍然有限。在这项研究中，我们证明了双向电泳（2-DE）提供了一种高分辨率和重复性的分析工具。因此，它已被应用于表征PTMs，包括糖基化，磷酸化，氧化和亚硝基化。检测小鼠心脏20S蛋白酶体及其相关蛋白的PTMs。我们的2-DE分析显示了超过25个20S复合物（17个亚基），表明心脏蛋白酶体有多个修饰亚基。质谱法支持2-DE后特异性PTM位点的鉴定。这些PTMs包括磷酸化和氧化。大多数PTMs发生在低化学计量，需要富集以提高检测灵敏度。总之，我们的研究支持2-DE作为分析20S蛋白酶体PTMs的核心工具。本研究中使用的方法证明了它们在绘制心脏组织20S蛋白酶体的PTMs方面的应用，这些方法适用于其他样品和生物学条件。
Post-translational modifications (PTMs) serve as key regulatory mechanisms for 20S proteasome functions. Alterations in 20S PTMs have been previously observed with changes in modified protein degradation patterns and altered cellular phenotypes. Despite decades of investigation, our knowledge pertaining to the various PTMs of 20S complexes and their biological significance remain limited. In this investigation, we show that two-dimensional electrophoresis (2-DE) offers an analytical tool with high resolution and reproducibility. Accordingly, it has been applied for the characterization of PTMs including glycosylation, phosphorylation, oxidation, and nitrosylation. The PTMs of murine cardiac 20S proteasomes and their associating proteins were examined. Our 2-DE analyses displayed over 25 spots for the 20S complexes (17 subunits), indicating multiply modified subunits of cardiac proteasomes. The identification of specific PTM sites subsequent to 2-DE were supported by mass spectrometry. These PTMs included phosphorylation and oxidation. Most of the PTMs occurred in low stoichiometry and required enrichment to enhance the detection sensitivity. In conclusion, our studies support 2-DE as a central tool in the analyses of 20S proteasome PTMs. The approaches utilized in this investigation demonstrate their application in mapping the PTMs of the 20S proteasomes in cardiac tissue, which are applicable to other samples and biological conditions.
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影响因子：
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