Proteomic mapping of the human mitochondrial intermembrane space in live cells via ratiometric APEX tagging.
Proteomic mapping of the human mitochondrial intermembrane space in live cells via ratiometric APEX tagging.
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DOI：
10.1016/j.molcel.2014.06.003
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发表时间：
2014-07-17
期刊：
影响因子：
16
通讯作者：
Ting AY
中科院分区：
文献类型：
--
作者：
Hung V;Zou P;Rhee HW;Udeshi ND;Cracan V;Svinkina T;Carr SA;Mootha VK;Ting AY
获取亚细胞区域的完整蛋白质清单是一项挑战，这常常限制我们对细胞功能的理解，特别是对于那些无法纯化、因此传统蛋白质组学分析无法触及的区域。我们最近开发了一种用一种工程化过氧化物酶（APEX）绘制活细胞蛋白质组图谱的方法，当应用于膜结合区室时，该方法无需细胞器纯化；然而，当应用于允许APEX产生的自由基逸出的无边界区域时，它缺乏特异性。在此，我们将APEX技术与一种基于稳定同位素标记细胞培养（SILAC）的比率标记策略相结合，以大幅减少不需要的背景并实现纳米级空间分辨率。这被应用于绘制线粒体膜间隙（IMS）的蛋白质组图谱，该区域可与细胞质自由交换小分子。我们所得到的包含127种蛋白质的IMS蛋白质组具有>94%的特异性，其中包括9种新的线粒体蛋白质。这种方法将使科学家能够绘制以前无法触及的细胞区域的蛋白质组图谱。
Obtaining complete protein inventories for subcellular regions is a challenge that often limits our understanding of cellular function, especially for regions that are impossible to purify and are therefore inaccessible to traditional proteomic analysis. We recently developed a method to map proteomes in living cells with an engineered peroxidase (APEX) that bypasses the need for organellar purification when applied to membrane-bound compartments; however, it lacked specificity when applied to unbounded regions that allow APEX-generated radicals to escape. Here, we combine APEX technology with a SILAC-based ratiometric tagging strategy to substantially reduce unwanted background and achieve nanometer spatial resolution. This is applied to map the proteome of the mitochondrial intermembrane space (IMS), which can freely exchange small molecules with the cytosol. Our IMS proteome of 127 proteins has >94% specificity and includes nine novel mitochondrial proteins. This approach will enable scientists to map proteomes of cellular regions that were previously inaccessible.