Defining Proximity Proteome of Histone Modifications by Antibody-mediated Protein A-APEX2 Labeling.
Defining Proximity Proteome of Histone Modifications by Antibody-mediated Protein A-APEX2 Labeling.
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DOI：
10.1016/j.gpb.2021.09.003
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发表时间：
2022-02
影响因子：
9.5
通讯作者：
Gan, Haiyun
中科院分区：
文献类型：
--
作者：
Li, Xinran;Zhou, Jiaqi;Zhao, Wenjuan;Wen, Qing;Wang, Weijie;Peng, Huipai;Gao, Yuan;Bouchonville, Kelly J.;Offer, Steven M.;Chan, Kuiming;Wang, Zhiquan;Li, Nan;Gan, Haiyun
由混杂酶（例如 APEX2）催化的邻近标记已成为表征多蛋白复合物和蛋白质-蛋白质相互作用的强大方法。然而，当前的方法依赖于外源融合蛋白的表达，并且不能应用于识别翻译后修饰蛋白周围的蛋白。为了解决这一限制，我们开发了一种新方法，通过抗体介导的蛋白 A-抗坏血酸过氧化物酶 2 (pA-APEX2) 标记 (AMAPEX) 来标记感兴趣的近端蛋白。在此方法中，修饰的蛋白质被特定抗体原位结合，然后束缚 pA-APEX2 融合蛋白。 APEX2 的激活会用生物素标记附近的蛋白质；然后使用链霉亲和素珠纯化生物素化的蛋白质并通过质谱法进行鉴定。我们通过分析组蛋白修饰的近端蛋白（包括 H3K27me3、H3K9me3、H3K4me3、H4K5ac 和 H4K12ac）证明了这种方法的实用性，并通过已发表的 ChIP-seq 分析和核小体免疫沉淀验证这些已识别蛋白与诱饵蛋白的共定位。总体而言，AMAPEX 是一种识别与修饰组蛋白最接近的蛋白质的有效方法。
Proximity labeling catalyzed by promiscuous enzymes, such as APEX2, has emerged as a powerful approach to characterize multiprotein complexes and protein–protein interactions. However, current methods depend on the expression of exogenous fusion proteins and cannot be applied to identify proteins surrounding post-translationally modified proteins. To address this limitation, we developed a new method to label proximal proteins of interest by antibody-mediated protein A-ascorbate peroxidase 2 (pA-APEX2) labeling (AMAPEX). In this method, a modified protein is bound in situ by a specific antibody, which then tethers a pA-APEX2 fusion protein. Activation of APEX2 labels the nearby proteins with biotin; the biotinylated proteins are then purified using streptavidin beads and identified by mass spectrometry. We demonstrated the utility of this approach by profiling the proximal proteins of histone modifications including H3K27me3, H3K9me3, H3K4me3, H4K5ac, and H4K12ac, as well as verifying the co-localization of these identified proteins with bait proteins by published ChIP-seq analysis and nucleosome immunoprecipitation. Overall, AMAPEX is an efficient method to identify proteins that are proximal to modified histones.
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影响因子：
48
作者：
Myers SA;Wright J;Peckner R;Kalish BT;Zhang F;Carr SA
通讯作者：
Carr SA
影响因子：
48
作者：
Gao XD;Tu LC;Mir A;Rodriguez T;Ding Y;Leszyk J;Dekker J;Shaffer SA;Zhu LJ;Wolfe SA;Sontheimer EJ
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Sontheimer EJ
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Ting, Alice Y.
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48
作者：
Bar DZ;Atkatsh K;Tavarez U;Erdos MR;Gruenbaum Y;Collins FS
通讯作者：
Collins FS
影响因子：
14.9
作者：
Deutsch EW;Csordas A;Sun Z;Jarnuczak A;Perez-Riverol Y;Ternent T;Campbell DS;Bernal-Llinares M;Okuda S;Kawano S;Moritz RL;Carver JJ;Wang M;Ishihama Y;Bandeira N;Hermjakob H;Vizcaíno JA
通讯作者：
Vizcaíno JA