Department of Chemistry, National Tsing Hua University, 101 Section 2, Kuang Fu Road, Hsinchu, 30013, Taiwan (Republic of China.
Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, 101 Section 2, Kuang Fu Road, Hsinchu, 30013, Taiwan (Republic of China.
Chem Asian J. 2020 Nov 2;15(21):3416-3420. doi: 10.1002/asia.202001049. Epub 2020 Sep 29.
The long-term stability of affinity-based protein labeling probes is crucial to obtain reproducible protein labeling results. However, highly stable probes generally suffer from low protein labeling efficiency and pose significant challenges when labeling low abundance native proteins in living cells. In this paper, we report that protein labeling probes based on an ortho-difluorophenyl ester reactive module exhibit long-term stability in DMSO stock solution and aqueous buffer, yet they can undergo rapid and selective labeling of native proteins. This novel electrophile can be customized with a wide range of different protein ligands and is particularly well-suited for the labeling and imaging of transmembrane proteins. With this probe design, the identity and relative levels of basal and hypoxia-induced transmembrane carbonic anhydrases were revealed by live cell imaging and in-gel fluorescence analysis. We believe that the extension of this difluorophenyl ester reactive module would allow for the specific labeling of various endogenous membrane proteins, facilitating in-depth studies of their distribution and functions in biological processes.
基于亲和力的蛋白质标记探针的长期稳定性对于获得可重复的蛋白质标记结果至关重要。然而，高稳定性的探针通常具有较低的蛋白质标记效率，并且在标记活细胞中低丰度的天然蛋白质时会带来重大挑战。在本文中，我们报告了基于邻-二氟苯酯反应性模块的蛋白质标记探针在 DMSO 储备溶液和水性缓冲液中具有长期稳定性，但它们可以快速且选择性地标记天然蛋白质。这种新型亲电试剂可以与广泛的不同蛋白质配体定制，并特别适合于跨膜蛋白的标记和成像。使用这种探针设计，通过活细胞成像和胶内荧光分析揭示了基础状态和缺氧诱导的跨膜碳酸酐酶的身份和相对水平。我们相信，这种邻-二氟苯酯反应性模块的扩展将允许各种内源性膜蛋白的特异性标记，从而促进对它们在生物过程中的分布和功能的深入研究。
