Department of Chemistry and Liver Cancer Institute of Zhongshan Hospital, Fudan University, Shanghai 200032, China.
Institutes of Biomedical Sciences and NHC Key Laboratory of Glycoconjugates Research, Fudan University, Shanghai 200032, China.
Anal Chem. 2024 Sep 10;96(36):14448-14455. doi: 10.1021/acs.analchem.4c02271. Epub 2024 Aug 27.
Cell surface proteins participate in many important biological processes, such as cell-to-cell interaction, signal transduction, cell adhesion, and protein transportation. In-depth study of the cell surface protein group is of great significance. Nevertheless, detection and analysis of the surfaceome remain a significant challenge due to their low abundance and hydrophobicity. Herein, we reported an ultrafast and chemoselective labeling method using our newly developed trifunctional probe, the OPA-S-S-alkyne, which labeled cell surface lysine residues, and then established a novel cell surfaceome profiling approach. According to our experimental results, the OPA-S-S-alkyne probe can react extremely fast with living cells, labeling cells in only 1 min, while traditional NHS (labeling cell surface lysine with -hydroxysuccinimide ester probe) and CSC (labeling cell surface glycan with hydrazide biotin probe) methods normally take longer time of more than 30 min and 1 h, respectively. Taking advantage of this ultrafast property of the method, we highlight the utility of this method by exploring the temporal dynamic changes of surfaceome upon EGF stimulation in living Hela cells and reported "early" and "late" EGF-regulated cell surface proteins, which are difficult to be distinguished by the current cell surface profiling approaches.
细胞表面蛋白参与许多重要的生物学过程，如细胞间相互作用、信号转导、细胞黏附和蛋白质运输。深入研究细胞表面蛋白组具有重要意义。然而，由于其丰度低和疏水性，细胞表面组的检测和分析仍然是一个重大挑战。在此，我们报道了一种使用我们新开发的三功能探针 OPA-S-S-alkyne 的超快和选择性化学标记方法，该探针标记细胞表面赖氨酸残基，然后建立了一种新的细胞表面组谱分析方法。根据我们的实验结果，OPA-S-S-alkyne 探针可以与活细胞快速反应，仅需 1 分钟即可标记细胞，而传统的 NHS（用 -羟基琥珀酰亚胺酯探针标记细胞表面赖氨酸）和 CSC（用酰肼生物素探针标记细胞表面聚糖）方法通常需要 30 多分钟和 1 小时的时间。利用该方法的超快特性，我们通过探索 EGF 刺激对活 Hela 细胞表面组的时间动态变化，突出了该方法的实用性，并报告了“早期”和“晚期”EGF 调节的细胞表面蛋白，这是目前的细胞表面分析方法难以区分的。
