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Rapid and Selective Labeling of Endogenous Transmembrane Proteins in Living Cells with a Difluorophenyl Ester Affinity‐Based Probe
Author(s) -
Chan HsinJu,
Lin XinHui,
Fan SyuanYun,
Ru Hwu Jih,
Tan KuiThong
Publication year - 2020
Publication title -
chemistry – an asian journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.18
H-Index - 106
eISSN - 1861-471X
pISSN - 1861-4728
DOI - 10.1002/asia.202001049
Subject(s) - transmembrane protein , chemistry , membrane protein , transmembrane domain , biochemistry , affinity labeling , integral membrane protein , fluorescence , target protein , endogeny , biophysics , membrane , biology , receptor , physics , quantum mechanics , gene
Abstract The long‐term stability of affinity‐based protein labeling probes is crucial to obtain reproducible protein labeling results. However, highly stable probes generally suffer from low protein labeling efficiency and pose significant challenges when labeling low abundance native proteins in living cells. In this paper, we report that protein labeling probes based on an ortho‐difluorophenyl ester reactive module exhibit long‐term stability in DMSO stock solution and aqueous buffer, yet they can undergo rapid and selective labeling of native proteins. This novel electrophile can be customized with a wide range of different protein ligands and is particularly well‐suited for the labeling and imaging of transmembrane proteins. With this probe design, the identity and relative levels of basal and hypoxia‐induced transmembrane carbonic anhydrases were revealed by live cell imaging and in‐gel fluorescence analysis. We believe that the extension of this difluorophenyl ester reactive module would allow for the specific labeling of various endogenous membrane proteins, facilitating in‐depth studies of their distribution and functions in biological processes.