College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing 100871, China.
State Key Laboratory of Proteomics, National Center for Protein Sciences, Beijing Proteome Research Center, Beijing Institute of Lifeomics, Beijing 102206, China.
Cell Chem Biol. 2020 Jul 16;27(7):858-865.e8. doi: 10.1016/j.chembiol.2020.05.006. Epub 2020 May 28.
The engineered ascorbate peroxidase (APEX) is a powerful tool for the proximity-dependent labeling of proteins and RNAs in live cells. Although widely use in mammalian cells, APEX applications in microorganisms have been hampered by the poor labeling efficiency of its biotin-phenol (BP) substrate. In this study, we sought to address this challenge by designing and screening a panel of alkyne-functionalized substrates. Our best probe, Alk-Ph, substantially improves APEX-labeling efficiency in intact yeast cells, as it is more cell wall-permeant than BP. Through a combination of protein-centric and peptide-centric chemoproteomic experiments, we have identified 165 proteins with a specificity of 94% in the yeast mitochondrial matrix. In addition, we have demonstrated that Alk-Ph is useful for proximity-dependent RNA labeling in yeast, thus expanding the scope of APEX-seq. We envision that this improved APEX-labeling strategy would set the stage for the large-scale mapping of spatial proteome and transcriptome in yeast.
工程化的抗坏血酸过氧化物酶（APEX）是一种强大的工具，可用于在活细胞中进行蛋白质和 RNA 的近距离依赖标记。尽管在哺乳动物细胞中得到了广泛应用，但由于其生物素-苯酚（BP）底物的标记效率较差，APEX 在微生物中的应用受到了阻碍。在这项研究中，我们通过设计和筛选一组炔基功能化的底物来解决这一挑战。我们的最佳探针 Alk-Ph 显著提高了完整酵母细胞中的 APEX 标记效率，因为它比 BP 更能穿透细胞壁。通过蛋白质中心和肽中心化学蛋白质组学实验的结合，我们在酵母线粒体基质中鉴定出了 165 种具有 94%特异性的蛋白质。此外，我们还证明了 Alk-Ph 可用于酵母中依赖于近距离的 RNA 标记，从而扩展了 APEX-seq 的范围。我们设想，这种改进的 APEX 标记策略将为在酵母中大规模绘制空间蛋白质组和转录组图谱奠定基础。
