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 cryogenic electron microscopy (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. Here, we present our online buffer exchange-native MS method combined with Direct Mass Technology (OBE-nMS+DMT) which facilitates high-throughput analysis and guides sample preparation. We applied this method to optimize the GPR119-Gs complex sample prior to cryo-EM analysis, leading to a 3.51 Å resolution structure from only 396 movies collected on a 200 kV Glacios. This study suggests that the OBE-nMS+DMT method emerges as a powerful tool for prescreening sample conditions in cryo-EM studies of GPCRs and other membrane protein complexes.

中文翻译：

---

用于冷冻电镜结构测定的 G 蛋白偶联受体复合物的天然质谱预筛选


G 蛋白偶联受体 （GPCR） 是在人类健康和疾病中起关键作用的必需跨膜蛋白。了解它们的原子级分子结构和构象状态对于推进药物开发至关重要。单颗粒低温电子显微镜 （cryo-EM） 的最新突破将 GPCR 的结构生物学推向了一个新时代。然而，由于稳定性差和高动态性，制备合适的 GPCR 样品及其复合物用于冷冻电镜分析仍然具有挑战性。在这里，我们介绍了我们的在线缓冲液交换非变性 MS 方法与直接质谱技术 （OBE-nMS+DMT） 相结合，该方法有助于高通量分析并指导样品制备。在冷冻电镜分析之前，我们应用这种方法来优化 GPR119-Gs 复合物样品，从而在 200 kV Glacios 上仅收集 396 部电影就获得了 3.51 Å 分辨率的结构。这项研究表明，OBE-nMS+DMT 方法已成为 GPCR 和其他膜蛋白复合物的冷冻电镜研究中预筛选样品条件的强大工具。