Chromatographic phospholipid trapping for automated H/D 1 exchange mass spectrometry analysis of membrane protein-lipid 2 assemblies

36 Lipid interactions modulate the function, folding, structure, and organization of membrane proteins. 37 Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has emerged as a useful tool to understand 38 the structural dynamics of these proteins within lipid environments. Lipids, however, have proven 39 problematic for HDX-MS analysis of membrane-embedded proteins, due to their presence impairing 40 proteolytic digestion, causing liquid chromatography column fouling, ion suppression, and/or mass spectral 41 overlap. Here, we describe the integration of a chromatographic phospholipid trap column into the HDX-42 MS apparatus to enable online sample delipidation prior to protease digestion of deuterium labeled protein-43 lipid assemblies. We demonstrate the utility of this method on membrane scaffold protein lipid nanodisc – 44 both empty and loaded with the ~115 kDa transmembrane protein AcrB – proving efficient and automated 45 phospholipid capture with minimal D-to-H back-exchange, peptide carry-over, and with minimal protein 46 loss. Our results provide insights into the efficiency of phospholipid capture by ZrO 2 -coated and TiO 2 47 beads, and describe how solution conditions can be optimized to maximize the performance of our online, 48 but also the existing offline, delipidation workflows for HDX-MS. We envision that this HDX-MS method 49 will significantly ease membrane protein analysis, allowing to better interrogate their dynamics in artificial 50 lipid bilayers or even cell membranes. 51 52 53

36. 脂质相互作用调节膜蛋白的功能、折叠、结构和组织。 37. 氢/氘交换质谱（HDX - MS）已成为了解脂质环境中这些蛋白质结构动力学的有用工具。 38. 39. 然而，由于脂质的存在会妨碍蛋白水解消化，导致液相色谱柱污染、离子抑制和/或质谱重叠，脂质已被证明对膜嵌入蛋白的HDX - MS分析存在问题。 40. 41. 42. 在此，我们描述了将色谱磷脂捕获柱整合到HDX - MS仪器中，以便在对氘标记的蛋白质 - 脂质复合物进行蛋白酶消化之前对样品进行在线脱脂。 43. 44. 我们在膜支架蛋白脂质纳米圆盘（包括空载的以及负载约115 kDa跨膜蛋白AcrB的）上证明了该方法的实用性，证明了其能高效且自动地捕获磷脂，同时具有最小的D - H反向交换、肽残留以及最小的蛋白质损失。 45. 46. 我们的结果深入了解了ZrO₂涂层和TiO₂珠对磷脂的捕获效率，并描述了如何优化溶液条件以最大限度地提高我们在线以及现有的离线HDX - MS脱脂工作流程的性能。 47. 48. 49. 我们设想这种HDX - MS方法将显著简化膜蛋白分析，使其能够更好地探究它们在人工脂质双层甚至细胞膜中的动力学。 50. 51. 52. 53.