In vivo mapping of tissue- and subcellular-specific proteomes in Caenorhabditis elegans.
In vivo mapping of tissue- and subcellular-specific proteomes in Caenorhabditis elegans.
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DOI：
10.1126/sciadv.1602426
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发表时间：
2017-05
期刊：
影响因子：
13.6
通讯作者：
Bennett EJ
中科院分区：
文献类型：
--
作者：
Reinke AW;Mak R;Troemel ER;Bennett EJ
新方法能够无偏地确定活动物体内的组织和亚细胞特异性蛋白质位置。多细胞生物体由具有不同功能的组织组成，需要专门的蛋白质组。为了定义活动物的蛋白质组与组织和亚细胞分辨率，我们采用了本地化的蛋白质组学技术用于多细胞模式生物秀丽隐杆线虫。这种方法结合了抗坏血酸过氧化物酶（APX）的组织特异性和位置特异性表达，这使得体内基于邻近的蛋白质标记和定量蛋白质组学能够识别组织和亚细胞限制性蛋白质组。我们鉴定并定位了3000多个来自C.在肠、表皮、体壁肌肉或咽肌的细胞核或细胞质中表达APX的线虫。我们还鉴定了几百种蛋白质，它们特异性地定位于所分析的四种组织之一或特异性地定位于细胞质或细胞核。这种方法导致在识别蛋白质与以前的特点定位和那些不知道本地化的细胞核或细胞质。此外，我们证实了组织和亚细胞特异性定位的一个子集的确定蛋白质使用绿色荧光蛋白标记和荧光显微镜，验证我们在体内的接近为基础的蛋白质组学技术。总之，这些结果证明了一种新的方法，使组织和亚细胞特异性识别和定量的蛋白质在一个活的动物。
New approach enables unbiased determination of tissue- and subcellular-specific protein location within a live animal. Multicellular organisms are composed of tissues that have distinct functions requiring specialized proteomes. To define the proteome of a live animal with tissue and subcellular resolution, we adapted a localized proteomics technology for use in the multicellular model organism Caenorhabditis elegans. This approach couples tissue- and location-specific expression of the enzyme ascorbate peroxidase (APX), which enables proximity-based protein labeling in vivo, and quantitative proteomics to identify tissue- and subcellular-restricted proteomes. We identified and localized more than 3000 proteins from strains of C. elegans expressing APX in either the nucleus or cytoplasm of the intestine, epidermis, body wall muscle, or pharyngeal muscle. We also identified several hundred proteins that were specifically localized to one of the four tissues analyzed or specifically localized to the cytoplasm or the nucleus. This approach resulted in the identification both of proteins with previously characterized localizations and of those not known to localize to the nucleus or cytoplasm. Further, we confirmed the tissue- and subcellular-specific localization of a subset of identified proteins using green fluorescent protein tagging and fluorescence microscopy, validating our in vivo proximity-based proteomics technique. Together, these results demonstrate a new approach that enables the tissue- and subcellular-specific identification and quantification of proteins within a live animal.