Proteome analysis of human nuclear insoluble fractions
Proteome analysis of human nuclear insoluble fractions
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DOI：
10.1111/j.1365-2443.2009.01324.x
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发表时间：
2009-08-01
期刊：
影响因子：
2.1
通讯作者：
Shibahara, Kei-ichi
中科院分区：
文献类型：
--
作者：
Takata, Hideaki;Nishijima, Hitoshi;Shibahara, Kei-ichi
间期核是一个高度有序的细胞器。关于可能存在什么复杂的机制来解释原子核的这些性质，我们知之甚少。同样未解决的是，是否一些建筑组件可能促进功能性核内区室或更高级的染色质结构的形成。作为解决这些问题的第一步，我们进行了深入的蛋白质组分析的人HeLa-S3细胞的核不溶性组分制备的两种不同的方法：高盐/去污剂/核酸酶抗性馏分和锂3，5-二碘水杨酸/核酸酶抗性馏分。通过液相色谱电喷雾串联质谱法分析各组分的蛋白质，分别从每个组分中鉴定出333和330种蛋白质。在不溶性核蛋白，我们确定了37个迄今未知的或功能不明的蛋白质。在这些蛋白质中经常发现RNA识别基序、WD 40重复序列、HEAT重复序列和SAP结构域。选定的蛋白质，包括DEK蛋白和SON蛋白的亚细胞分布，证明了它们与核不溶性物质的新关联，证实了我们基于MS的分析。这项研究建立了一个全面的人类细胞核不溶性蛋白质的目录。在我们的研究中确定的蛋白质的进一步功能分析将显着提高我们的理解间期核的动态组织。
The interphase nucleus is a highly ordered and compartmentalized organelle. Little is known regarding what elaborate mechanisms might exist to explain these properties of the nucleus. Also unresolved is whether some architectural components might facilitate the formation of functional intranuclear compartments or higher order chromatin structure. As the first step to address these questions, we performed an in-depth proteome analysis of nuclear insoluble fractions of human HeLa-S3 cells prepared by two different approaches: a high-salt/detergent/nuclease-resistant fraction and a lithium 3,5-diiodosalicylate/nuclease-resistant fraction. Proteins of the fractions were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry, identifying 333 and 330 proteins from each fraction respectively. Among the insoluble nuclear proteins, we identified 37 hitherto unknown or functionally uncharacterized proteins. The RNA recognition motif, WD40 repeats, HEAT repeats and the SAP domain were often found in these identified proteins. The subcellular distribution of selected proteins, including DEK protein and SON protein, demonstrated their novel associations with nuclear insoluble materials, corroborating our MS-based analysis. This study establishes a comprehensive catalog of the nuclear insoluble proteins in human cells. Further functional analysis of the proteins identified in our study will significantly improve our understanding of the dynamic organization of the interphase nucleus.