G protein-coupled receptors (GPCRs) are essential transmembrane proteins playing key roles in human health and disease. Understanding their atomic-level molecular structure and conformational states is imperative for advancing drug development. Recent breakthroughs in single-particle cryo-EM have propelled the structural biology of GPCRs into a new era. Nevertheless, the preparation of suitable GPCR samples and their complexes for cryo-EM analysis remains challenging due to their poor stability and highly dynamic nature. Although standard analytical methods such as SDS-PAGE and SEC indicated acceptable quality for most samples, this did not always translate into promising cryo-EM results.
In this study, we present a two-step prescreening workflow—online buffer exchange-native mass spectrometry combined with Direct Mass Technology (OBE-nMS+DMT)—to enhance sample preparation of the human GPCR119-Gs protein complex for cryo-EM structure determination. The OBE-nMS+DMT workflow includes: 1) OBE-nMS for subunits/subcomplexes analysis, 2) DMT for integrative complex determination. OBE-nMS using (lauryldimethylamine-N-oxide) LDAO in the mobile phase allows confirmation of protein sequences and detection of post-translational modifications (PTMs), though LDAO as a zwitter-ionic detergent may dissociate complexes. Therefore, to characterize intact complexes, n-dodecyl-β-maltoside (DDM) is preferred as the non-ionic detergent for membrane protein analysis by nMS. However, the heterogeneous nature of GPCR-G protein complexes often produces complex, unresolved native MS spectra in DDM. To overcome this, Direct Mass Technology enables simultaneous mass-to-charge and charge measurements for resolving traditionally unresolvable biomolecules.
We applied this method to optimize the GPCR119-Gs complex, adjusting ligand type and concentration, as well as detergent selection, before cryo-EM analysis. The optimized sample yielded a 3.51 Å resolution structure from only 396 movies collected on a 200 kV Glacios microscope. This study demonstrates that the OBE-nMS+DMT workflow offers comprehensive insights by revealing critical differences in composition, stoichiometry, and homogeneity among samples, thereby enhancing cryo-EM outcomes for challenging GPCR complexes.