Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging.
Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging.
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DOI：
10.1126/science.1230593
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发表时间：
2013-03-15
期刊：
影响因子：
--
通讯作者：
Ting AY
中科院分区：
文献类型：
--
作者：
Rhee HW;Zou P;Udeshi ND;Martell JD;Mootha VK;Carr SA;Ting AY
显微镜和质谱（MS）是互补的技术：前者提供活细胞中的时空信息，但仅针对少数重组蛋白，而后者可以同时检测数千种内源性蛋白，但仅在裂解样品中。在这里，我们介绍的技术，通过提供活细胞内内源性蛋白质的空间和时间分辨的蛋白质组图谱结合这些优势。该方法依赖于一种遗传靶向的过氧化物酶，它使附近的蛋白质生物素化，随后通过MS纯化和鉴定。我们使用这种方法鉴定了人类线粒体基质中的495种蛋白质，包括31种以前未与线粒体连接的蛋白质。标记是非常具体的，并区分内膜蛋白面对的基质与膜间空间（IMS）。几种蛋白质以前认为驻留在IMS或外膜，包括原卟啉原氧化酶，被重新分配到矩阵。活细胞过氧化物酶介导的蛋白质组图谱的特异性与其易用性相结合，为生物学家提供了一个强大的工具，了解活细胞的分子组成。
Microscopy and mass spectrometry (MS) are complementary techniques: the former provides spatiotemporal information in living cells, but only for a handful of recombinant proteins, while the latter can detect thousands of endogenous proteins simultaneously, but only in lysed samples. Here we introduce technology that combines these strengths by offering spatially- and temporally-resolved proteomic maps of endogenous proteins within living cells. The method relies on a genetically-targetable peroxidase enzyme that biotinylates nearby proteins, which are subsequently purified and identified by MS. We used this approach to identify 495 proteins within the human mitochondrial matrix, including 31 not previously linked to mitochondria. The labeling was exceptionally specific and distinguished between inner membrane proteins facing the matrix versus the intermembrane space (IMS). Several proteins previously thought to reside in the IMS or outer membrane, including protoporphyrinogen oxidase, were reassigned to the matrix. The specificity of live-cell peroxidase-mediated proteomic mapping combined with its ease of use offers biologists a powerful tool for understanding the molecular composition of living cells.
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影响因子：
4.7
作者：
Minamihata, Kosuke;Goto, Masahiro;Kamiya, Noriho
通讯作者：
Kamiya, Noriho
影响因子：
2.9
作者：
Rogers, Melanie S.;Hurtado-Guerrero, Ramon;McPherson, Michael J.
通讯作者：
McPherson, Michael J.
DOI：
10.1083/jcb.201112098
发表时间：
2012-03-19
期刊：
The Journal of cell biology
影响因子：
--
作者：
Roux KJ;Kim DI;Raida M;Burke B
通讯作者：
Burke B
影响因子：
56.9
作者：
Bendayan, M
通讯作者：
Bendayan, M
影响因子：
6.1
作者：
Mortensen, A;Skibsted, LH
通讯作者：
Skibsted, LH