Immobilizing the Endogenous 5-Hydroxytryptamine Transporter by Ligand-Directed and Proximity-Catalyzed Acyl Imidazole Chemistry for Affinity Chromatographic Analysis

The sensitivity, selectivity, and accuracy of immobilized protein-based methods are critically dependent on the strategies employed for protein immobilization. Compared with random immobilization approaches, site-specific covalent methods have emerged as promising alternatives, offering enhanced analytical performance. However, these methods typically require genetic modification of the target protein to incorporate a specific tag or prior purification of the protein, posing significant challenges for immobilizing endogenous proteins. Herein, we address these limitations by using the 5-hydroxytryptamine transporter (5-HTT) as a model system. We designed a probe by conjugating fluvoxamine─a specific ligand of 5-HTT─with either a fluorescent reporter or aminopropyl-modified silica gel through an alkyl linker containing an acyl imidazole moiety as the reactive group. By introducing ligand-directed acyl imidazole (LDAI) chemistry, we achieved selective labeling of 5-HTT in live cells and endogenous 5-HTT immobilization without purification in advance. Labeling experiments confirmed the high specificity and favorable kinetics of the LDAI ligands toward 5-HTT. Chromatographic analysis revealed significant improvements in selectivity (5.7–50.9-fold), resolution (1.2–3.0-fold), and sensitivity (1.0–12.0-fold) for analyzing canonical ligands when using immobilized endogenous 5-HTT, compared to His-tagged and Halo-tagged recombinant proteins. This approach, by substituting fluvoxamine with other ligands in the probe, can be generalized for the labeling and immobilization of a wide range of endogenous proteins, offering a versatile platform for advanced protein-based analytical techniques.