Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2.
Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2.
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DOI：
10.1038/nprot.2016.018
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发表时间：
2016-03
期刊：
影响因子：
14.8
通讯作者：
Ting AY
中科院分区：
文献类型：
--
作者：
Hung V;Udeshi ND;Lam SS;Loh KH;Cox KJ;Pedram K;Carr SA;Ting AY
该协议描述了一种方法，以获得空间分辨蛋白质组图的特定隔间内活的哺乳动物细胞。一种工程化的过氧化物酶APEX 2，在基因上靶向感兴趣的细胞区域。在将过氧化氢添加到预装载有生物素-苯酚底物的细胞中1分钟后，APEX 2产生共价标记近端内源性蛋白质的生物素-苯氧基自由基。然后裂解细胞，用链霉亲和素珠富集生物素化蛋白质，并通过质谱法鉴定。我们描述了一个适当的APEX 2融合构建，蛋白质组学样品制备，质谱数据采集和分析的一代。一个双态稳定同位素标记的氨基酸在细胞培养（SILAC）协议是用于蛋白质组学映射的膜封闭的细胞隔室，APEX 2产生的生物素-苯氧基自由基不能逃脱。对于开放的细胞区域的映射，我们反而使用“比率”三态SILAC协议的高空间特异性。蛋白质的同位素标记需要5-7个细胞倍增时间。生成生物素化的蛋白质组样品需要1天，获得质谱数据需要2-5天，分析数据以获得最终的蛋白质组列表需要1周。
This protocol describes a method to obtain spatially resolved proteomic maps of specific compartments within living mammalian cells. An engineered peroxidase, APEX2, is genetically targeted to a cellular region of interest. Upon the addition of hydrogen peroxide for 1 min to cells preloaded with a biotin-phenol substrate, APEX2 generates biotin-phenoxyl radicals that covalently tag proximal endogenous proteins. Cells are then lysed, and biotinylated proteins are enriched with streptavidin beads and identified by mass spectrometry. We describe the generation of an appropriate APEX2 fusion construct, proteomic sample preparation, and mass spectrometric data acquisition and analysis. A two-state stable isotope labeling by amino acids in cell culture (SILAC) protocol is used for proteomic mapping of membrane-enclosed cellular compartments from which APEX2-generated biotin-phenoxyl radicals cannot escape. For mapping of open cellular regions, we instead use a ‘ratiometric’ three-state SILAC protocol for high spatial specificity. Isotopic labeling of proteins takes 5–7 cell doublings. Generation of the biotinylated proteomic sample takes 1 d, acquiring the mass spectrometric data takes 2–5 d and analysis of the data to obtain the final proteomic list takes 1 week.