Characterization of the human COP9 signalosome complex using affinity purification and mass spectrometry.
Characterization of the human COP9 signalosome complex using affinity purification and mass spectrometry.
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DOI：
10.1021/pr800574c
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发表时间：
2008-11
影响因子：
4.4
通讯作者：
Huang L
中科院分区：
文献类型：
--
作者：
Fang L;Wang X;Yamoah K;Chen PL;Pan ZQ;Huang L
COP 9信号体（CSN）是一种多蛋白复合物，在各种真核生物的多种细胞和发育过程中起着关键作用。尽管它的重要性，目前的理解的生物学功能和调控机制的CSN复合物仍然非常有限。为了解开这些分子机制，我们已经进行了全面的蛋白质组学分析的人CSN复杂使用一种新的纯化方法和定量质谱。通过高亲和力链霉亲和素结合TEV切割洗脱，从表达N-末端HBTH标记的CSN 5亚基的稳定293细胞系中纯化人CSN复合物。纯化的CSN复合物的质谱分析揭示了其组成的身份以及CSN亚基的N-末端修饰和磷酸化。N-末端修饰确定了7个亚基，其中6个以前没有报道过，还确定了6个新的磷酸化位点。此外，我们应用新开发的MAP-SILAC和PAM-SILAC方法来破译人类CSN相互作用蛋白的动力学。共鉴定了52个推测的人CSN相互作用蛋白，其中大部分是首次报道。与PAM-SILAC结果相比，20种蛋白质被归类为稳定的相互作用物，而20种蛋白质被鉴定为动态的。这项工作提出了第一个全面的表征的人CSN复合物的质谱为基础的蛋白质组学方法，提供了有价值的信息，为进一步了解CSN复合物的结构和生物学功能。
The COP9 signalosome (CSN) is a multiprotein complex that plays a critical role in diverse cellular and developmental processes in various eukaryotic organisms. Despite of its significance, current understanding of the biological functions and regulatory mechanisms of the CSN complex is still very limited. To unravel these molecular mechanisms, we have performed a comprehensive proteomic analysis of the human CSN complex using a new purification method and quantitative mass spectrometry. Purification of the human CSN complex from a stable 293 cell line expressing N-terminal HBTH-tagged CSN5 subunit was achieved by high-affinity streptavidin binding with TEV cleavage elution. Mass spectrometric analysis of the purified CSN complex has revealed the identity of its composition as well as N-terminal modification and phosphorylation of the CSN subunits. N-terminal modifications were determined for seven subunits, six of which have not been reported previously, and six novel phosphorylation sites were also identified. Additionally, we have applied the newly developed MAP-SILAC and PAM-SILAC methods to decipher the dynamics of the human CSN interacting proteins. A total of 52 putative human CSN interacting proteins were identified, most of which are reported for the first time. In comparison to PAM-SILAC results, 20 proteins were classified as stable interactors, whereas 20 proteins were identified as dynamic ones. This work presents the first comprehensive characterization of the human CSN complex by mass spectrometry-based proteomic approach, providing valuable information for further understanding of CSN complex structure and biological functions.
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影响因子：
11.4
作者：
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通讯作者：
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