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

Lam

Chemistry An Asian Journal

et al. 2021

Self Cite

To date, various affinity-based protein labeling probes have been developed and applied in biological research to modify endogenous proteins in cell lysates and on the cell surface. However, the reactive groups on the labeling probes are also the cause of probe instability and nonselective labeling in a more complex environment, e. g., intracellular and in vivo. Here, we show that labeling probes composed of a sterically stabilized difluorophenyl pivalate can achieve efficient and selective labeling of endogenous proteins on the cell surface, inside living cells and in vivo. As compared with the existing protein labeling probes, probes with the difluorophenyl pivalate exhibit several advantages, including long-term stability in stock solutions, resistance to enzymatic hydrolysis and can be customized easily with diverse fluorophores and protein ligands. With this probe design, endogenous hypoxia biomarker in living cells and nude mice were successfully labeled and validated by in vivo, ex vivo, and immunohistochemistry imaging.

Section: Resultsmentioning

confidence: 81%

Improved Stabilities of Labeling Probes for the Selective Modification of Endogenous Proteins in Living Cells and In Vivo

Lam

Chemistry An Asian Journal

et al. 2021

Self Cite

“…To achieve efficient labeling of the cell surface hCA proteins, the probes were functionalized with the difluorophenyl ester reactive group, which was reported previously by our group (Figure S2). , The detailed synthetic schemes to prepare these labeling probes are described in the Supporting Information (Schemes S1–S5). By using MALDI-TOF and in-gel Western blot analysis, we confirmed that 1 can be labeled efficiently to the purified hCAI and transmembrane hCAIX from MCF7 cells, respectively (Figure S3).…”

Section: Resultsmentioning

confidence: 99%

Small-Molecule Modulated Affinity-Tunable Semisynthetic Protein Switches

Shao-jie

et al. 2022

ACS Sens.

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Engineered protein switches have been widely applied in cell-based protein sensors and point-of-care diagnosis for the rapid and simple analysis of a wide variety of proteins, metabolites, nucleic acids, and enzymatic activities. Currently, these protein switches are based on two main types of switching mechanisms to transduce the target binding event to a quantitative signal, through a change in the optical properties of fluorescent molecules and the activation of enzymatic activities. In this paper, we introduce a new affinity-tunable protein switch strategy in which the binding of a small-molecule target with the protein activates the streptavidin–biotin interaction to generate a readout signal. In the absence of a target, the biotinylated protein switch forms a closed conformation where the biotin is positioned in close proximity to the protein, imposing a large steric hindrance to prevent the effective binding with streptavidin. In the presence of the target molecule, this steric hindrance is removed, thereby exposing the biotin for streptavidin binding to produce strong fluorescent signals. With this modular sensing concept, various sulfonamide, methotrexate, and trimethoprim drugs can be selectively detected on the cell surface of native and genetically engineered cells using different fluorescent dyes and detection techniques.

“…1a), which have been used for covalent binding of ligands/inhibitors. 28–32 The emerging ligand-directed chemistry has been used to label endogenous proteins in living systems, including tosyl (LDT), 33–35 alkyloxyacyl imidazole (LDAI), 36–38 O -nitrobenzoxadiazole ( O -NBD), 39 dibromophenylbenzoate (LDBB), 40 difluorophenylbenzoate, 41 N -sulfonyl pyridone, 42 and N -acyl- N -alkyl sulfonamide (LDNASA) chemistry (Fig. 1a).…”

Section: Introductionmentioning

confidence: 99%

Ultrafast and selective labeling of endogenous proteins using affinity-based benzotriazole chemistry

et al. 2022