Characterization of Covalently-Bound Agonists of kappa-Opioid Receptors By Mass Spectrometric Analysis

摘要

Salvinorin A, the most potent naturally-occurring hallucinogen, has gained great attention since the kappa-Opioid Receptor (KOR) was identified as its principal molecular target (Roth et al, PNAS 2002). Previously, extensive efforts were made to understand how salvinorin A binds to and activates KOR. The current goal of the project was to design a series of ligands capable of covalently binding to KOR to further explore the ligand-receptor interactions at atomic level. For covalent attachment, we used chemically-reactive amino acids as nucleophiles. After the incubation of the live cells expressing KOR with the ligand(s), the receptor proteins were purified/separated by SDS-PAGE, and the band containing the KOR protein was in-gel digested with chymotrypsin. The eluted peptides were analyzed using an AB 4800 MALDI-TOF/TOF mass spectrometer, to locate the KOR-containing band. Using this technique, however, no modification site was detected. The digest was then acquired on a Bruker Ultraflex MALDI TOF/TOF, using HCCA as matrix. Spectra of digests from the protein with and without the attached ligand were compared in short mass regions in order to detect the shifted masses, and the selected ions were further analyzed by MALDI-MS/MS.