A quantitative proteomics analysis of subcellular proteome localization and changes induced by DNA damage.
A quantitative proteomics analysis of subcellular proteome localization and changes induced by DNA damage.
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DOI：
10.1074/mcp.m900429-mcp200
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发表时间：
2010-03
期刊：
影响因子：
--
通讯作者：
Lamond AI
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文献类型：
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作者：
Boisvert FM;Lam YW;Lamont D;Lamond AI
细胞生物学中的一个主要挑战是确定细胞内蛋白质的亚细胞分布，并表征蛋白质定位在不同细胞生长条件下以及响应应力和其他外部信号时如何变化。蛋白质定位通常通过显微镜或通过使用细胞分级分离结合蛋白质印迹技术来确定。这两种方法本质上都是低通量的，并且限于对已知组分的分析。在这里，我们使用基于质谱的蛋白质组学提供了一个公正的，定量的，高通量的方法来测量蛋白质组的亚细胞分布，称为“空间蛋白质组学”。空间蛋白质组学方法分析通过重组来自细胞的差异标记的亚细胞级分而产生的全细胞提取物，其中蛋白质已经用重同位素进行质量标记。在这里，这被用来测量HCT 116细胞中超过2,000种蛋白质在细胞质、细胞核和核仁之间的相对分布。数据显示，在稳态下，蛋白质组主要分配到特定的亚细胞位置，只有一小部分蛋白质均匀分布在两个或多个隔室之间。空间蛋白质组学还有助于蛋白质组范围内的比较蛋白质定位的变化，在响应范围广泛的生理和实验扰动，在这里显示的特征动态变化引起的蛋白质定位在细胞响应DNA损伤后，用依托泊苷治疗HCT 116细胞。发现DNA损伤导致蛋白酶体从细胞质中的抑制蛋白和组装伴侣解离，并重新定位为与细胞核中的蛋白酶体激活剂相关联。
A major challenge in cell biology is to identify the subcellular distribution of proteins within cells and to characterize how protein localization changes under different cell growth conditions and in response to stress and other external signals. Protein localization is usually determined either by microscopy or by using cell fractionation combined with protein blotting techniques. Both these approaches are intrinsically low throughput and limited to the analysis of known components. Here we use mass spectrometry-based proteomics to provide an unbiased, quantitative, and high throughput approach for measuring the subcellular distribution of the proteome, termed “spatial proteomics.” The spatial proteomics method analyzes a whole cell extract created by recombining differentially labeled subcellular fractions derived from cells in which proteins have been mass-labeled with heavy isotopes. This was used here to measure the relative distribution between cytoplasm, nucleus, and nucleolus of over 2,000 proteins in HCT116 cells. The data show that, at steady state, the proteome is predominantly partitioned into specific subcellular locations with only a minor subset of proteins equally distributed between two or more compartments. Spatial proteomics also facilitates a proteome-wide comparison of changes in protein localization in response to a wide range of physiological and experimental perturbations, shown here by characterizing dynamic changes in protein localization elicited during the cellular response to DNA damage following treatment of HCT116 cells with etoposide. DNA damage was found to cause dissociation of the proteasome from inhibitory proteins and assembly chaperones in the cytoplasm and relocation to associate with proteasome activators in the nucleus.