Catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS), implemented using model membranes (MMs), is a promising approach for the discovery of glycolipid ligands of glycan-binding proteins (GBPs). Picodiscs (PDs), which are lipid-transporting complexes composed of the human sphingolipid activator protein saposin A and phospholipids, have proven to be useful MMs for such studies. The present work compares the use of conventional (pre-loaded) PDs with passively loaded PDs (^PLPDs) for CaR-ESI-MS screening of glycolipids against cholera toxin B subunit homopentamer (CTB₅). The pre-loaded PDs were prepared from a mixture of purified glycolipid and phospholipid or a mixture of lipids extracted from tissue, while the ^PLPDs were prepared by incubating PDs containing only phospholipid with glycolipid-containing lipid mixtures in aqueous solution. Time-dependent changes in the composition of the ^PLPDs produced by incubation with glycomicelles of the ganglioside GM1 were monitored using collision-induced dissociation of the gaseous PD ions and from the extent of ganglioside binding to CTB₅ measured by ESI-MS. GM1 incorporation into PDs was evident within a few hours of incubation. At incubation times ≥ 10 days, GM1 binding to CTB₅ was indistinguishable from that observed with pre-loaded PDs produced directly from GM1 at the same concentration. Comparison of ganglioside binding to CTB₅ measured for pre-loaded PDs and ^PLPDs prepared from glycolipids extracted from pig and mouse brain revealed that the ^PLPDs allow for the detection of a greater number of ganglioside ligands. Together, the results of this study suggest ^PLPDs may have advantages over conventionally prepared PDs for screening glycolipids against GBPs using CaR-ESI-MS.

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使用模型膜（MM）实施的捕集和释放电喷雾电离质谱（CaR-ESI-MS），是发现聚糖结合蛋白（GBP）糖脂配体的一种有前途的方法。Picodiscs（PDs）是由人类鞘脂激活蛋白saposin A和磷脂组成的脂质转运复合物，已被证明是用于此类研究的MM。本工作比较了常规（预加载）PD和被动加载PD（^PL PD）在针对霍乱毒素B亚基均戊四烯（CTB ₅）的糖脂的CaR-ESI-MS筛选中的用途。预载的PD是由纯化的糖脂和磷脂的混合物或从组织中提取的脂质的混合物制备的，而^PL通过将仅包含磷脂的PD与含糖脂的脂质混合物在水溶液中孵育来制备PD。与神经节苷脂GM1的糖胶团一起温育产生的^PL PDs的组成随时间变化是通过气态PD离子的碰撞诱导离解和通过ESI-MS测量的神经节苷脂与CTB ₅结合的程度来监测的。在孵育的几个小时内就将GM1掺入了PD中是显而易见的。在≥10天的孵育时间中，与CTB ₅结合的GM1与直接由相同浓度的GM1直接产生的预加载PD所观察到的没有区别。神经节苷脂与CTB ₅结合的比较，用于预加载的PD和^PL从猪和小鼠大脑中提取的糖脂制备的PD揭示了^PL PD可以检测更多数量的神经节苷脂配体。总之，这项研究的结果表明，^PL CaPDs可能比常规制备的PDs具有更多优势，可以使用CaR-ESI-MS筛选针对GBPs的糖脂。

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