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首页> 外文期刊>Molecular & cellular proteomics: MCP >A quantitative proteomics analysis of subcellular proteome localization and changes induced by DNA damage.
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A quantitative proteomics analysis of subcellular proteome localization and changes induced by DNA damage.

机译：定量蛋白质组学分析的亚细胞蛋白质组定位和DNA损伤诱导的变化。

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摘要

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.
机译：细胞生物学的一个主要挑战是鉴定蛋白质在细胞内的亚细胞分布，并表征在不同细胞生长条件下以及对压力和其他外部信号的响应下蛋白质定位的变化。通常通过显微镜或通过结合蛋白质印迹技术的细胞分级来确定蛋白质的定位。这两种方法本质上都是低吞吐量，并且仅限于分析已知组件。在这里，我们使用基于质谱的蛋白质组学来提供一种无偏见，定量且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了一种全细胞提取物，该提取物是通过重组来自细胞的差异标记亚细胞部分而产生的，在细胞中蛋白质已被重同位素进行了质量标记。在此用于测量HCT116细胞中2,000多种蛋白质的细胞质，细胞核和核仁之间的相对分布。数据显示，在稳定状态下，蛋白质组主要被划分为特定的亚细胞位置，只有一小部分蛋白质均匀分布在两个或多个区室之间。空间蛋白质组学还促进了蛋白质组变化的蛋白质组变化比较，以响应各种生理和实验扰动，此处通过表征依托泊苷处理HCT116细胞后细胞对DNA损伤的细胞反应过程中引起的蛋白质局部变化的动态变化，来显示蛋白质组变化的变化。 。发现DNA损伤导致蛋白酶体与细胞质中的抑制蛋白和组装伴侣解离，并重新定位以与细胞核中的蛋白酶体激活剂结合。

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