Rapid and Selective Labeling of Endogenous Transmembrane Proteins in Living Cells with a Difluorophenyl Ester Affinity‐Based Probe

• Published in: Chemistry, an Asian journal Vol. 15; no. 21; pp. 3416 - 3420
• Main Authors: Chan, Hsin‐Ju, Lin, Xin‐Hui, Fan, Syuan‐Yun, Ru Hwu, Jih, Tan, Kui‐Thong
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 02.11.2020
• Subjects: Affinity; Biological activity; Cell Line, Tumor; Cells (biology); Chemistry; Difluorophenyl ester; Esters - chemical synthesis; Esters - chemistry; Fluorescence; Fluorescent Dyes - chemical synthesis; Fluorescent Dyes - chemistry; Fluorescent Imaging; Humans; Hydrocarbons, Fluorinated - chemical synthesis; Hydrocarbons, Fluorinated - chemistry; Hypoxia; Labeling; Membrane proteins; Membrane Proteins - analysis; Modules; Molecular Structure; Optical Imaging; Protein labeling; Proteins; Stability; Staining and Labeling; Fluorescent Imaging; Hypoxia; Difluorophenyl ester; Protein labeling; Membrane proteins
• DOI: 10.1002/asia.202001049
• ISSN: 1861-4728

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. Endogenous protein labeling: Affinity‐based protein labeling probes based on an ortho‐difluorophenyl ester reactive module exhibit long term stability in stock solution and aqueous buffer, yet they can undergo rapid and selective modification of native proteins in living cells.