Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling
Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling
复制标题
DOI：
10.1016/j.isci.2019.11.025
复制
发表时间：
2019-12-20
期刊：
影响因子：
5.8
通讯作者：
Ojida, Akio
中科院分区：
文献类型：
--
作者：
Tabata, Shigekazu;Jevtic, Marijo;Ojida, Akio
电子显微镜（EM）是一种能够在纳米分辨率下显示单个蛋白质的技术。然而，目前的EM蛋白质分析主要依赖于金颗粒偶联抗体的免疫标记，这受到抗体大尺寸的影响，无法精确检测生物样品中的蛋白质位置。在这里，我们开发了一种特定的化学标记方法，用于单分子水平的蛋白质EM检测。合理设计的α -螺旋肽标签和探针结构提供了一个互补的反应对，使标签的特异性半胱氨酸偶联成为可能。与免疫金标记相比，利用金纳米粒子偶联探针的化学标记方法对冷冻断裂复制品中高密度G蛋白偶联受体簇的标记效率和可检测性显著提高。此外，在超薄切片中，化学标记的空间分辨率显著高于抗体介导的标记。这些结果证明了化学标记方法在EM单蛋白可视化中的巨大优势。
Electron microscopy (EM) is a technology that enables visualization of single proteins at a nanometer resolution. However, current protein analysis by EM mainly relies on immunolabeling with gold-particle-conjugated antibodies, which is compromised by large size of antibody, precluding precise detection of protein location in biological samples. Here, we develop a specific chemical labeling method for EM detection of proteins at single-molecular level. Rational design of alpha-helical peptide tag and probe structure provided a complementary reaction pair that enabled specific cysteine conjugation of the tag. The developed chemical labeling with gold-nanoparticle-conjugated probe showed significantly higher labeling efficiency and detectability of high-density clusters of tag-fused G protein-coupled receptors in freeze-fracture replicas compared with immunogold labeling. Furthermore, in ultrathin sections, the spatial resolution of the chemical labeling was significantly higher than that of antibody-mediated labeling. These results demonstrate substantial advantages of the chemical labeling approach for single protein visualization by EM.