Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling.

Munetsugu Kido,Naoki Zenmyo,Harumi Harada,Hirokazu Fuchida,Shigekazu Tabata,Ryuichi Shigemoto,Itaru Hamachi,Akio Ojida,Kazushi Tani,Makoto Itakura,Nobutaka Kurashige,Marijo Jevtic,Shohei Uchinomiya

doi:10.1016/j.isci.2019.11.025

Abstract

SummaryElectron 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 α-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.

Highlights

Electron microscopy (EM) is a powerful technique for detecting localization and distribution of a protein of interest (POI) in biological specimens at a nanometer resolution
EM detection using the tag-probe pair was successfully applied to determine the localization of B2R receptor on cell surface with a high labeling specificity
EM detection methods for in-cell proteins using genetically encoded peroxidases have been developed in recent years (Shu et al, 2011; Martell et al, 2012; Hainfeld and Powell, 2000)

Summary

Introduction

Electron microscopy (EM) is a powerful technique for detecting localization and distribution of a protein of interest (POI) in biological specimens at a nanometer resolution. Immunogold labeling usually suffers from inherent problems, which are mainly associated with the large molecular size of antibody The use of bulky primary and secondary antibodies locates gold particles 20–30 nm away from POI (Fijita et al, 2007), precluding precise detection of protein localization in biological samples. The large molecular size of antibody hinders its penetration into biological specimens, making it challenging to quantitatively compare POI localization along tissue depth (Masugi-Tokita and Shigemoto, 2007). Chemical protein labeling methods using small molecular probe could potentially overcome these problems. Despite their extensive use in fluorescence imaging, application to single-protein detection by EM is yet to be reported

Methods

Results

Conclusion