研究细胞中蛋白质组的空间分布对于阐明诸多细胞生物学中的分子机理有重要意义。近年来，以APEX（The engineered ascorbate peroxidase）为代表的邻近化学标记技术迅速发展，实现了活细胞环境内特定空间区域蛋白质和RNA的邻近标记，避免传统物理分离细胞器方法造成的假阳性。

APEX技术利用过氧化物酶催化探针底物生物素-苯酚（BP），产生高活性自由基中间体，对周围的蛋白质和RNA进行共价加成反应，实现空间特异性标记^[1]。APEX可应用于多个生物学体系^[2-5]，但传统BP底物分子体积大、溶解度低，限制了它在酵母、细菌等微生物领域中的应用。

北京大学邹鹏研究员课题组设计并成功合成新一代应用于微生物体系的APEX探针底物——炔基苯酚（Alkyne-Phenol，Alk-Ph）^[6]。

和传统底物BP相比，有如下优势：

• 分子体积更小（MW: 217.27）；
• 细胞壁和细胞膜穿透性能更好，水溶液中溶解度更高；
• 良好空间特异性和更高覆盖度，使APEX标记效率提高一个数量级（图1）。

图1 The higher efficiency of Alk-Ph labeling in yeast^[6]. (D) Analysis of the mitochondrial specificity of APEX2 labeling. Left: proteins with prior mitochondrial annotations in the entire S. cerevisiae proteome (from Uniprot database). Middle: our mitochondrial matrix proteome labeled with Alk-Ph. Right: previous mitochondrial matrix proteome labeled with BP. (E) Comparison of the APEX2-labeling coverage with Alk-Ph and BP probes.

邹鹏研究员课题组已成功在活酵母细胞中使用Alk-Ph对蛋白质（图2）和RNA（图3）进行邻近标记，并利用定量蛋白质组和转录组学数据证明了该探针具有良好的空间特异性和更高的覆盖度。

Alk-Ph探针还能揭示APEX在蛋白上的修饰位点，为深入研究蛋白拓扑结构和构象提供技术支持。

图2 APEX Labeling of Yeast Subcellular Proteome^[6]. （A）Scheme of APEX-mediated labeling with Alk-Ph probe in yeast. （B）Confocal fluorescence images of W303 yeast cells expressing Su9-APEX2-eGFP, Su9-eGFP, and NLS-APEX2-eGFP.

图3 APEX2-Mediated Alk-Ph Labeling of the Mitochondrial Transcriptome^[6]. (A) Scheme of RNA labeling in yeast. (B) Real-time-PCR analysis of mitochondrial RNA enrichment elative to the cytoplasmic RNA markers, ACT1 and THD3.

邹鹏研究员科研成果转化，百灵威独家提供应用于微生物体系的蛋白质和RNA邻近标记探针——Alk-Ph。

Alkyne-phenol, 95%

1694495-59-4

9186096
10 MG 50 MG

参考文献

1. Rhee, H.-W., Zou, P., Udeshi, N.D., Martell, J.D., Mootha, V.K., Carr, S.A., and Ting, A.Y. (2013). Proteomic Mapping of Mitochondria in Living Cells via Spatially Restricted Enzymatic Tagging. Science 339, 1328-1331.
2. Hung, V., Lam, S.S., Udeshi, N.D., Svinkina, T., Guzman, G., Mootha, V.K., Carr, S.A., and Ting, A.Y. (2017). Proteomic mapping of cytosol-facing outer mitochondrial and ER membranes in living human cells by proximity biotinylation. eLife 6, e24463.
3. Hung, V., Udeshi, N.D., Lam, S.S., Loh, K.H., Cox, K.J., Pedram, K., Carr, S.A., and Ting, A.Y. (2016). Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2. Nat Protoc 11, 456-475.
4. Hung, V., Zou, P., Rhee, H.-W., Udeshi, Namrata D., Cracan, V., Svinkina, T., Carr, Steven A., Mootha, Vamsi K., and Ting, Alice Y. (2014). Proteomic Mapping of the Human Mitochondrial Intermembrane Space in Live Cells via Ratiometric APEX Tagging. Mol Cell 55, 332-341.
5. Paek, J., Kalocsay, M., Staus, D.P., Wingler, L., Pascolutti, R., Paulo, J.A., Gygi, S.P., and Kruse, A.C. (2017). Multidimensional Tracking of GPCR Signaling via Peroxidase-Catalyzed Proximity Labeling. Cell 169, 338-349.
6. Li, Y., Tian, C., Liu, K., Zhou, Y., Yang, J., and Zou, P. (2020). A Clickable APEX Probe for Proximity-Dependent Proteomic Profiling in Yeast. Cell Chem Biol 27, 858-865.e858.

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