Institut Laue-Langevin, 71 avenue des Martyrs, CS 20156, 38042 Grenoble, France; École doctorale de Physique, Université Grenoble Alpes, 38400 Saint-Martin-d'Héres, France.
Institut Laue-Langevin, 71 avenue des Martyrs, CS 20156, 38042 Grenoble, France.
J Colloid Interface Sci. 2023 Sep;645:870-881. doi: 10.1016/j.jcis.2023.04.135. Epub 2023 May 5.
Cellular membranes are complex systems that consist of hundreds of different lipid species. Their investigation often relies on simple bilayer models including few synthetic lipid species. Glycerophospholipids (GPLs) extracted from cells are a valuable resource to produce advanced models of biological membranes. Here, we present the optimisation of a method previously reported by our team for the extraction and purification of various GPL mixtures from Pichia pastoris. The implementation of an additional purification step by High Performance Liquid Chromatography-Evaporative Light Scattering Detector (HPLC-ELSD) enabled for a better separation of the GPL mixtures from the neutral lipid fraction that includes sterols, and also allowed for the GPLs to be purified according to their different polar headgroups. Pure GPL mixtures at significantly high yields were produced through this approach. For this study, we utilised phoshatidylcholine (PC), phosphatidylserine (PS) and phosphatidylglycerol (PG) mixtures. These exhibit a single composition of the polar head, i.e., PC, PS or PG, but contain several molecular species consisting of acyl chains of varying length and unsaturation, which were determined by Gas Chromatography (GC). The lipid mixtures were produced both in their hydrogenous (H) and deuterated (D) versions and were used to form lipid bilayers both on solid substrates and as vesicles in solution. The supported lipid bilayers were characterised by quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR), whereas the vesicles by small angle X-ray (SAXS) and neutron scattering (SANS). Our results show that despite differences in the acyl chain composition, the hydrogenous and deuterated extracts produced bilayers with very comparable structures, which makes them valuable to design experiments involving selective deuteration with techniques such as NMR, neutron scattering or infrared spectroscopy.
细胞膜是由数百种不同脂质组成的复杂系统。对它们的研究通常依赖于包含少数几种合成脂质的简单双层模型。从细胞中提取的甘油磷脂（GPL）是构建生物膜先进模型的宝贵资源。在这里，我们对我们团队之前报道的从毕赤酵母中提取和纯化各种 GPL 混合物的方法进行了优化。通过高效液相色谱-蒸发光散射检测器（HPLC-ELSD）增加一个额外的纯化步骤，可以更好地将 GPL 混合物与包括固醇在内的中性脂质部分分离，也可以根据不同的极性头基对 GPL 进行纯化。通过这种方法可以获得产量非常高的纯 GPL 混合物。在这项研究中，我们使用了磷脂酰胆碱（PC）、磷脂酰丝氨酸（PS）和磷脂酰甘油（PG）混合物。这些混合物的极性头基均为单一组成，即 PC、PS 或 PG，但包含几种由不同长度和不饱和度的酰链组成的分子种类，这些酰链的组成通过气相色谱（GC）确定。这些脂质混合物以其氢化物（H）和氘化物（D）版本生成，并用于在固体基底上形成脂质双层以及在溶液中形成囊泡。通过石英晶体微天平耗散监测（QCM-D）和中子反射率（NR）对支撑脂质双层进行了表征，通过小角 X 射线（SAXS）和中子散射（SANS）对囊泡进行了表征。我们的结果表明，尽管酰链组成存在差异，但氢化物和氘化物提取物形成的双层具有非常相似的结构，这使得它们对于设计涉及 NMR、中子散射或红外光谱等技术的选择性氘化实验非常有价值。
