Institute of Drug Metabolism and Pharmaceutical Analysis, Research Center for Clinical Pharmacy, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China; Key Laboratory of Intelligent Pharmacy and Individualized Therapy of Huzhou, Department of Pharmacy, Changxing People's Hospital, Huzhou, 313100, China.
Institute of Drug Metabolism and Pharmaceutical Analysis, Research Center for Clinical Pharmacy, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China; Institute of Translational Medicine, Zhejiang University, Hangzhou, 310029, China.
Anal Chim Acta. 2024 Nov 22;1330:343296. doi: 10.1016/j.aca.2024.343296. Epub 2024 Oct 1.
Cell-surface proteins play key roles in the communication between external stimuli and internal signaling. As protein types and expression levels vary in different cells, in-situ visualization of the whole surface proteome (surfaceome) may facilitate the study of their functions in homeostasis maintenance or response to environmental changes (e.g., drug treatment). However, there lacks easily-prepared and universal labeling probes to visualize them in living cells.
We designed and synthesized a small-molecule fluorescent probe, SRB-NHS, for one-step labeling of surfaceome. Live-cell imaging results exhibited the plasma membrane localization of the fluorescent signal from SRB-NHS and SDS-PAGE/fluorescence scanning results confirmed the covalent labeling of proteins by SRB-NHS, indicating the suitability of SRB-NHS for surfaceome labeling towards different cell lines.
Upon labeling by SRB-NHS, the cellular internalization of surfaceome was studied under different stimuli (e.g., nutritional deprivation, drug treatments). Intriguingly, specific monitoring of the interaction between antibody drugs and related cell-surface targets can be achieved when the probe is used in combination with fluorescently labeled antibodies and imaged via Förster resonance energy transfer (FRET), offering a new method compatible with various cell lines to monitor the surfaceome or a specific drug-target interaction in situ.
细胞表面蛋白在外部刺激与内部信号转导之间的通讯中发挥着关键作用。由于不同细胞中的蛋白质类型和表达水平不同，因此原位可视化整个表面蛋白质组（表面组）可能有助于研究它们在维持内稳态或响应环境变化（例如药物治疗）中的功能。然而，缺乏易于制备和通用的标记探针来在活细胞中可视化它们。
我们设计并合成了一种小分子荧光探针 SRB-NHS，用于表面组的一步标记。活细胞成像结果显示了来自 SRB-NHS 的荧光信号的质膜定位，并且 SDS-PAGE/荧光扫描结果证实了 SRB-NHS 对蛋白质的共价标记，表明 SRB-NHS 适合用于不同细胞系的表面组标记。
在用 SRB-NHS 标记后，在不同刺激（例如营养剥夺、药物处理）下研究了表面组的细胞内化。有趣的是，当将探针与荧光标记的抗体结合使用并通过Förster 共振能量转移（FRET）成像时，可以实现对抗体药物与相关细胞表面靶标之间相互作用的特异性监测，为原位监测表面组或特定药物-靶标相互作用提供了一种与各种细胞系兼容的新方法。
