APEX Proximity Labeling as a Versatile Tool for Biological Research.,Biochemistry

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APEX Proximity Labeling as a Versatile Tool for Biological Research.
Biochemistry ( IF 2.9 ) Pub Date : 2019-12-03 , DOI: 10.1021/acs.biochem.9b00791
Thanh My Thi Nguyen ₁ , Junhyung Kim ₁ , Thi Tram Doan ₁ , Min-Woo Lee ₁ , Mihye Lee ₁

Affiliation

Most proteins are specifically localized in membrane-encapsulated organelles or non-membrane-bound compartments. The subcellular localization of proteins facilitates their functions and integration into functional networks; therefore, protein localization is tightly regulated in diverse biological contexts. However, protein localization has been mainly analyzed through immunohistochemistry or the fractionation of subcellular compartments, each of which has major drawbacks. Immunohistochemistry can examine only a handful of proteins at a time, and fractionation inevitably relies on the lysis of cells, which disrupts native cellular conditions. Recently, an engineered ascorbate peroxidase (APEX)-based proximity labeling technique combined with mass spectrometry was developed, which allows for temporally and spatially resolved proteomic mapping. In the presence of H2O2, engineered APEX oxidizes biotin-phenols into biotin-phenoxyl radicals, and these short-lived radicals biotinylate electron-rich amino acids within a radius of several nanometers. Biotinylated proteins are subsequently enriched by streptavidin and identified by mass spectrometry. This permits the sensitive and efficient labeling of proximal proteins around locally expressed APEX. Through the targeted expression of APEX in the subcellular region of interest, proteomic profiling of submitochondrial spaces, the outer mitochondrial membrane, the endoplasmic reticulum (ER)-mitochondrial contact, and the ER membrane has been performed. Furthermore, this method has been modified to define interaction networks in the vicinity of target proteins and has also been applied to analyze the spatial transcriptome. In this Perspective, we provide an outline of this newly developed technique and discuss its potential applications to address diverse biological questions.

中文翻译：

APEX邻近标签可作为生物研究的多功能工具。

大多数蛋白质特异性地定位在膜囊化的细胞器或非膜结合的区室中。蛋白质的亚细胞定位促进了它们的功能和整合到功能网络中。因此，蛋白质的定位在不同的生物学环境中受到严格的调控。但是，蛋白质定位主要是通过免疫组织化学或亚细胞区室分离来分析的，每种方法都有主要缺点。免疫组织化学一次只能检查少量蛋白质，而分级分离不可避免地依赖于细胞裂解，这会破坏天然细胞状况。最近，开发了一种基于工程抗坏血酸过氧化物酶（APEX）的邻近标记技术，并结合了质谱技术，该技术可在时间和空间上解析蛋白质组图谱。在过氧化氢的存在下，经过工程设计的APEX将生物素酚氧化为生物素-苯氧基自由基，这些短寿命的自由基将几纳米半径内的富含电子的氨基酸生物素化。随后通过抗生蛋白链菌素富集生物素化的蛋白质，并通过质谱鉴定。这允许在局部表达的APEX周围敏感和有效地标记近端蛋白质。通过在感兴趣的亚细胞区域中有针对性地表达APEX，进行了线粒体空间，线粒体外膜，内质网（ER）-线粒体接触和ER膜的蛋白质组分析。此外，已对该方法进行了修改，以在目标蛋白附近定义相互作用网络，并已应用于分析空间转录组。从这个角度来看，

更新日期：2019-12-04

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