Systematic characterization of nuclear Proteome during apoptosis - A quantitative proteomic study by differential extraction and stable isotope labeling
Systematic characterization of nuclear Proteome during apoptosis - A quantitative proteomic study by differential extraction and stable isotope labeling
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DOI：
10.1074/mcp.m500162-mcp200
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发表时间：
2006-06-01
影响因子：
7
通讯作者：
Han, David K.
中科院分区：
文献类型：
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作者：
Hwang, Sun-Il;Lundgren, Deborah H.;Han, David K.
核蛋白质组的鉴定和鉴定对于详细了解真核细胞中的多种信号事件具有重要意义。为了实现这一目标，我们通过使用三种缓冲条件连续提取具有不同物理化学性质的核蛋白来广泛地表征人T白血病细胞的核蛋白质组。这项大规模的蛋白质组学研究还测试了细胞培养中氨基酸稳定同位素标记(SILAC)揭示细胞凋亡过程中数量变化的可行性和技术挑战。分析从幼稚细胞和凋亡细胞中提取的三个核部分的蛋白质，产生了780,530个MS/MS光谱，用于使用SEQUEST算法进行数据库搜索。这一分析导致了1,174种可能的核蛋白的鉴定和定量。一些已知的与细胞凋亡有关的核蛋白以及一些未知的新蛋白被鉴定和定量。与基于SILAC的定量结果一致，对细胞核、线粒体和两个细胞器中的一些相关蛋白的免疫荧光染色显示，在细胞凋亡过程中，线粒体动态地重新聚集到核想象中。
Identification and characterization of the nuclear proteome is important for detailed understanding of multiple signaling events in eukaryotic cells. Toward this goal, we extensively characterized the nuclear proteome of human T leukemia cells by sequential extraction of nuclear proteins with different physicochemical properties using three buffer conditions. This large scale proteomic study also tested the feasibility and technical challenges associated with stable isotope labeling by amino acids in cell culture (SILAC) to uncover quantitative changes during apoptosis. Analyzing proteins from three nuclear fractions extracted from naive and apoptotic cells generated 780,530 MS/MS spectra that were used for database searching using the SEQUEST algorithm. This analysis resulted in the identification and quantification of 1,174 putative nuclear proteins. A number of known nuclear proteins involved in apoptosis as well as novel proteins not known to be part of the nuclear apoptotic machinery were identified and quantified. Consistent with SILAC-based quantifications, immunofluorescence staining of nucleus, mitochondria, and some associated proteins from both organelles revealed a dynamic recruitment of mitochondria into nuclear imaginations during apoptosis.