Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry.
Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry.
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DOI：
10.1021/pr800468j
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发表时间：
2008-10
影响因子：
4.4
通讯作者：
Kaiser, Peter
中科院分区：
文献类型：
--
作者：
Meierhofer, David;Wang, Xiaorong;Huang, Lan;Kaiser, Peter
泛素化通过标记蛋白质进行降解来调节细胞过程，但也作为一种调节性的非蛋白水解翻译后修饰发挥作用。蛋白质组范围内的策略，以监测泛素化的变化概况是重要的，以获得洞察泛素化的各种细胞功能。在这里，我们描述了稳定的细胞系表达的串联hexahistidine-生物素标签（HB-标签）融合到泛素的两步纯化的泛素化的蛋白质组在完全变性条件下。使用这种方法，我们确定了669泛素化的蛋白质从HeLa细胞，包括44个精确的泛素附着位点的基板和所有七个可能的泛素链连接类型。为了探索泛素化的动力学响应于泛素/蛋白酶体途径的扰动，我们结合泛素分析与定量质谱使用稳定同位素标记与细胞培养中的氨基酸（SILAC）策略。我们比较了未处理的细胞和用蛋白酶体抑制剂MG 132处理的细胞，以鉴定靶向蛋白酶体降解的泛素化蛋白。鉴定了许多蛋白酶体底物。此外，定量的方法使我们能够比较蛋白酶体靶向不同的泛素链拓扑结构在体内。这里描述的工具和策略可以应用于检测响应于生长条件和细胞应激的各种变化的泛素化动力学的变化，并将有助于我们对泛素/蛋白酶体系统的理解。
Ubiquitination regulates a host of cellular processes by labeling proteins for degradation, but also by functioning as a regulatory, nonproteolytic posttranslational modification. Proteome-wide strategies to monitor changes in ubiquitination profiles are important to obtain insight into the various cellular functions of ubiquitination. Here we describe generation of stable cell lines expressing a tandem hexahistidine-biotin tag (HB-tag) fused to ubiquitin for two-step purification of the ubiquitinated proteome under fully denaturing conditions. Using this approach we identified 669 ubiquitinated proteins from HeLa cells, including 44 precise ubiquitin attachment sites on substrates and all seven possible ubiquitin chain-linkage types. To probe the dynamics of ubiquitination in response to perturbation of the ubiquitin/proteasome pathway, we combined ubiquitin profiling with quantitative mass spectrometry using the stable isotope labeling with amino acids in cell culture (SILAC) strategy. We compared untreated cells and cells treated with the proteasome inhibitor MG132 to identify ubiquitinated proteins that are targeted to the proteasome for degradation. A number of proteasome substrates were identified. In addition, the quantitative approach allowed us to compare proteasome targeting by different ubiquitin chain topologies in vivo. The tools and strategies described here can be applied to detect changes in ubiquitination dynamics in response to various changes in growth conditions and cellular stress and will contribute to our understanding of the ubiquitin/proteasome system.
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影响因子：
2.3
作者：
Groothuis TA;Dantuma NP;Neefjes J;Salomons FA
通讯作者：
Salomons FA
影响因子：
3.4
作者：
Kirkpatrick, DS;Weldon, SF;Gandolfi, AJ
通讯作者：
Gandolfi, AJ
影响因子：
4.8
作者：
Ong, SE;Foster, LJ;Mann, M
通讯作者：
Mann, M
影响因子：
16
作者：
Garcia-Higuera, I;Taniguchi, T;D'Andrea, AD
通讯作者：
D'Andrea, AD
影响因子：
21.3
作者：
Kirkpatrick, Donald S.;Hathaway, Nathaniel A.;Gygi, Steven P.
通讯作者：
Gygi, Steven P.