Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Sci Adv. 2017 May 10;3(5):e1602426. doi: 10.1126/sciadv.1602426. eCollection 2017 May.
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 . 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 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.
多细胞生物由具有不同功能的组织组成，这些组织需要专门的蛋白质组。为了定义具有组织和亚细胞分辨率的活体动物的蛋白质组，我们对一种适用于多细胞模式生物的局部蛋白质组学技术进行了改编。这种方法结合了组织和位置特异性表达抗坏血酸过氧化物酶（APX），这使得能够在体内进行基于邻近的蛋白质标记，并进行定量蛋白质组学以鉴定组织和亚细胞受限的蛋白质组。我们从表达 APX 的菌株中鉴定和定位了超过 3000 种蛋白质，这些菌株的 APX 分别位于肠、表皮、体壁肌肉或咽肌的细胞核或细胞质中。我们还鉴定了数百种专门定位于这四种组织之一或专门定位于细胞质或细胞核的蛋白质。这种方法不仅鉴定了具有先前描述的定位的蛋白质，还鉴定了那些以前不知道定位于细胞核或细胞质的蛋白质。此外，我们使用绿色荧光蛋白标记和荧光显微镜验证了鉴定出的一部分蛋白质的组织和亚细胞特异性定位，验证了我们的体内邻近蛋白质组学技术。总之，这些结果展示了一种新的方法，能够在活体动物中鉴定和定量具有组织和亚细胞特异性的蛋白质。
