Evaluation of Drug Responses to Human β(2)AR Using Native Mass Spectrometry

[Image: see text] We aimed to develop a platform to rapidly investigate the responses of agonists and antagonists to G-protein-coupled receptors (GPCRs) using native mass spectrometry (MS). We successfully observed the ligand-bound human β(2) adrenergic receptor (hβ(2)AR); however, it was challenging to quantitatively discuss drug efficacy from MS data alone. Since ligand-bound GPCRs are stabilized by the Gα subunit of G proteins on the membrane, mini-G(s) and nanobody80 (Nb80) that can mimic the Gα interface of the GPCR were utilized. Ternary complexes of hβ(2)AR, ligand, and mini-G(s) or Nb80 were prepared and subjected to native MS. We found a strong correlation between the hβ(2)AR–mini-G(s) or −Nb80 complex ratio observed in the mass spectra and agonist/antagonist efficacy obtained using a cell-based assay. This method does not require radioisotope labeling and would be applicable to the analysis of other GPCRs, facilitating the characterization of candidate compounds as GPCR agonists and antagonists.