CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Anal Chem. 2021 Mar 16;93(10):4542-4551. doi: 10.1021/acs.analchem.0c04970. Epub 2021 Mar 4.
Cell surface is the primary site for sensing extracellular stimuli. The knowledge of the transient changes on the surfaceome upon a perturbation is very important as the initial changed proteins could be driving molecules for some phenotype. In this study, we report a fast cell surface labeling strategy based on peroxidase-mediated oxidative tyrosine coupling strategy, enabling efficient and selective cell surface labeling within seconds. With a labeling time of 1 min, 2684 proteins, including 1370 (51%) cell surface-annotated proteins (cell surface/plasma membrane/extracellular), 732 transmembrane proteins, and 81 cluster of differentiation antigens, were identified from HeLa cells. By comparison with the negative control experiment using quantitative proteomics, 500 (68%) out of the 731 significantly enriched proteins (-value < 0.05, ≥2-fold) in positive experimental samples were cell surface-annotated proteins. Finally, this technology was applied to track the dynamic changes of the surfaceome upon insulin stimulation at two time points (5 min and 2 h) in HepG2 cells. Thirty-two proteins, including INSR, CTNNB1, TFRC, IGF2R, and SORT1, were found to be significantly regulated (-value < 0.01, ≥1.5-fold) after insulin exposure by different mechanisms. We envision that this technique could be a powerful tool to analyze the transient changes of the surfaceome with a good time resolution and to delineate the temporal and spatial regulation of cellular signaling.
细胞表面是感知细胞外刺激的主要部位。了解扰动后表面组的瞬时变化非常重要，因为最初变化的蛋白质可能是某些表型的驱动分子。在这项研究中，我们报告了一种基于过氧化物酶介导的氧化酪氨酸偶联策略的快速细胞表面标记策略，能够在几秒钟内实现高效和选择性的细胞表面标记。在 1 分钟的标记时间内，从 HeLa 细胞中鉴定出 2684 种蛋白质，包括 1370 种（51%）细胞表面/质膜/细胞外标记的蛋白质、732 种跨膜蛋白质和 81 种分化抗原。通过与使用定量蛋白质组学的阴性对照实验进行比较，在阳性实验样品中显著富集的 731 种蛋白质中有 500 种（68%）（-值<0.05，≥2 倍）为细胞表面标记的蛋白质。最后，该技术被应用于跟踪 HepG2 细胞中胰岛素刺激后两个时间点（5 分钟和 2 小时）表面组的动态变化。在胰岛素暴露后，通过不同的机制发现 32 种蛋白质（包括 INSR、CTNNB1、TFRC、IGF2R 和 SORT1）发生了显著调节（-值<0.01，≥1.5 倍）。我们设想，该技术可以成为一种强大的工具，用于以良好的时间分辨率分析表面组的瞬时变化，并描绘细胞信号的时空调节。
