Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, U.K.
Global Research Technologies, Novo Nordisk A/S, Novo Nordisk Park, Måløv 2760, Denmark.
J Am Chem Soc. 2021 Mar 24;143(11):4085-4089. doi: 10.1021/jacs.0c11837. Epub 2021 Mar 12.
The use of mass spectrometry to investigate proteins is now well established and provides invaluable information for both soluble and membrane protein assemblies. Maintaining transient noncovalent interactions under physiological conditions, however, remains challenging. Here, using nanoscale electrospray ionization emitters, we establish conditions that enable mass spectrometry of two G protein-coupled receptors (GPCR) from buffers containing high concentrations of sodium ions. For the Class A GPCR, the adenosine 2A receptor, we observe ligand-induced changes to sodium binding of the receptor at the level of individual sodium ions. We find that antagonists promote sodium binding while agonists attenuate sodium binding. These findings are in line with high-resolution X-ray crystallography wherein only inactive conformations retain sodium ions in allosteric binding pockets. For the glucagon receptor (a Class B GPCR) we observed enhanced ligand binding in electrospray buffers containing high concentrations of sodium, as opposed to ammonium acetate buffers. A combination of native and -omics mass spectrometry revealed the presence of a lipophilic negative allosteric modulator. These experiments highlight the advantages of implementing native mass spectrometry, from electrospray buffers containing high concentrations of physiologically relevant salts, to inform on allosteric ions or ligands with the potential to define their roles on GPCR function.
利用质谱法研究蛋白质现在已经得到了很好的确立，并为可溶性和膜蛋白组装体提供了非常有价值的信息。然而，在生理条件下维持瞬时非共价相互作用仍然具有挑战性。在这里，我们使用纳米级电喷雾发射器，建立了在含有高浓度钠离子的缓冲液中对两种 G 蛋白偶联受体（GPCR）进行质谱分析的条件。对于 A 类 GPCR，即腺苷 2A 受体，我们观察到配体诱导的单个钠离子水平上的受体钠离子结合的变化。我们发现拮抗剂促进钠离子结合，而激动剂则减弱钠离子结合。这些发现与高分辨率 X 射线晶体学一致，其中只有非活性构象在变构结合口袋中保留钠离子。对于胰高血糖素受体（B 类 GPCR），我们观察到在含有高浓度钠离子的电喷雾缓冲液中，与含有醋酸铵的缓冲液相比，配体结合增强。结合天然和组学质谱法揭示了亲脂性负变构调节剂的存在。这些实验强调了从含有生理相关盐的电喷雾缓冲液中实施天然质谱法的优势，以了解变构离子或配体，它们有可能定义它们在 GPCR 功能中的作用。
