Chromatographic Phospholipid Trapping for Automated H/D Exchange Mass Spectrometry of Membrane Protein-Lipid Assemblies.
Chromatographic Phospholipid Trapping for Automated H/D Exchange Mass Spectrometry of Membrane Protein-Lipid Assemblies.
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DOI：
10.1021/acs.analchem.2c04876
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发表时间：
2023-02-07
影响因子：
7.4
通讯作者：
Reading, Eamonn
中科院分区：
文献类型：
--
作者：
Hammerschmid, Dietmar;Calvaresi, Valeria;Bailey, Chloe;Lewis, Benjamin Russell;Politis, Argyris;Morris, Michael;Denbigh, Laetitia;Anderson, Malcolm;Reading, Eamonn
脂类相互作用调节膜蛋白的功能、折叠、结构和组织。氢/氢交换质谱仪(HDX-MS)已成为了解这些蛋白质在脂质环境中的结构动力学的有用工具。然而，脂类在膜包埋蛋白质的HDX-MS分析中已被证明是有问题的，因为它们的存在阻碍了蛋白质的消化，导致了液相色谱柱污染、离子抑制和/或质谱图重叠。在这里，我们描述了将色谱磷脂捕捉柱集成到HDX-MS设备中，以使在线样品在酶消化氚标记的蛋白质-脂组件之前能够脱脂。我们展示了这种方法在膜支架蛋白质-脂纳米盘上的应用--无论是空的还是装载了∼115 kDa跨膜蛋白AcrB的--证明了高效和自动化的磷脂捕获，以及最小的D-to-H反向交换、多肽携带和蛋白质损失。我们的研究结果为我们深入了解了包裹了氧化锆和二氧化钛的微珠捕获磷脂的效率，并描述了如何优化溶液条件，使我们的在线工作流程和现有的脱脂脱脂工作流程在HDX-MS中的性能最大化。我们预计，这种HDX-MS方法将大大简化膜蛋白分析，允许更好地询问它们在人工脂双层甚至天然细胞膜中的动力学。
Lipid interactions modulate the function, folding, structure, and organization of membrane proteins. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has emerged as a useful tool to understand the structural dynamics of these proteins within lipid environments. Lipids, however, have proven problematic for HDX-MS analysis of membrane-embedded proteins due to their presence of impairing proteolytic digestion, causing liquid chromatography column fouling, ion suppression, and/or mass spectral overlap. Herein, we describe the integration of a chromatographic phospholipid trap column into the HDX-MS apparatus to enable online sample delipidation prior to protease digestion of deuterium-labeled protein–lipid assemblies. We demonstrate the utility of this method on membrane scaffold protein–lipid nanodisc—both empty and loaded with the ∼115 kDa transmembrane protein AcrB—proving efficient and automated phospholipid capture with minimal D-to-H back-exchange, peptide carry-over, and protein loss. Our results provide insights into the efficiency of phospholipid capture by ZrO2-coated and TiO2 beads and describe how solution conditions can be optimized to maximize not only the performance of our online but also the existing offline, delipidation workflows for HDX-MS. We envision that this HDX-MS method will significantly ease membrane protein analysis, allowing to better interrogate their dynamics in artificial lipid bilayers or even native cell membranes.
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DOI：
10.1007/s13361-012-0377-z
发表时间：
2012-06
影响因子：
3.2
作者：
Iacob, Roxana E.;Engen, John R.
通讯作者：
Engen, John R.
影响因子：
48
作者：
Masson, Glenn R.;Burke, John E.;Rand, Kasper D.
通讯作者：
Rand, Kasper D.
影响因子：
4.4
作者：
Kyono, Yutaka;Sugiyama, Naoyuki;Ishihama, Yasushi
通讯作者：
Ishihama, Yasushi
影响因子：
7
作者：
Larsen, MR;Thingholm, TE;Jorgensen, TJD
通讯作者：
Jorgensen, TJD
影响因子：
--
作者：
Hasui, K;Murata, F
通讯作者：
Murata, F