RESUMEN
Rapid access to appropriately functionalized probes is crucial in chemical labeling approaches to target identification studies. We designed and synthesized clickable gold-nanoparticles as generic probe precursors that enable (1) one-step ligand derivatization by click chemistry, and (2) facile photoaffinity labeling application. Using cholesterol as a model ligand, we successfully demonstrated the utility of the ligand-clicked probe in photoaffinity labeling of endogenously expressed oxysterol-binding protein (OSBP) in cell lysate.
Asunto(s)
Nanopartículas del Metal/química , Etiquetas de Fotoafinidad/química , Alquinos/química , Animales , Azidas/química , Anhidrasa Carbónica II/química , Bovinos , Colesterol/análogos & derivados , Química Clic , Oro/química , Ligandos , Etiquetas de Fotoafinidad/síntesis química , Etiquetas de Fotoafinidad/efectos de la radiación , Receptores de Esteroides/química , Rayos UltravioletaRESUMEN
Alkyne-labelled proteins are generated as key intermediates in the chemical probe-based approaches to proteomics analysis. Their efficient and selective detection and isolation is an important problem. We designed and synthesized azide-functionalized gold nanoparticles as new clickable capture reagents to streamline click chemistry-mediated capture, enrichment and release of the alkyne-labelled proteins in one-pot to expedite the post-labelling analysis. Because hydrophobic surface functionalities are known to render gold nanoparticles poorly water-dispersible, hydrophilic PEG linkers with two different lengths were explored to confer colloidal stability to the clickable capture reagents. We demonstrated the ability of the capture reagents to conjugate the alkyne containing proteins at a nanomolar concentration via click chemistry, which can be immediately followed by their enrichment and elution. Furthermore, a bifunctional clickable capture reagent bearing sulforhodamine and azide groups was shown to conveniently attach a fluorophore to the alkyne-labelled protein upon click capture, which facilitated their rapid detection in the gel analysis.