Researchers choose different methods of making giant unilamellar vesicles to satisfy different constraints of their experimental designs. A challenge that arises when researchers use a variety of methods is that each method may produce vesicles with a different average lipid ratio, even if all experiments use lipids from a common stock mixture. Here, we use mass spectrometry to investigate ratios of lipids in vesicle solutions made by five common methods: electroformation on indium tin oxide slides, electroformation on platinum wires, gentle hydration, emulsion transfer, and extrusion. We made vesicles from either five-component or binary mixtures of lipids chosen to span a wide range of physical properties: di(18:1)PC, di(16:0)PC, di(18:1)PG, di(12:0)PE, and cholesterol. For a mixture of all five of these lipids, ITO electroformation, Pt electroformation, gentle hydration, and extrusion methods result in only minor shifts in lipid ratios (≤5 mol %) relative to a common stock solution. In contrast, emulsion transfer results in ∼80% less cholesterol than expected from the stock solution, which is counterbalanced by a surprising overabundance of saturated PC-lipid relative to all other phospholipids. Experiments using binary mixtures of saturated and unsaturated PC-lipids and cholesterol largely support results from the five-component mixture. In general, our results imply that experiments that increment lipid ratios in small steps will produce data that are highly sensitive to the technique used and to sample-to-sample variations. For example, sample-to-sample variations are ∼±2 mol % for five-component vesicles produced by a single technique. In contrast, experiments that explore larger increments in lipid ratio or that seek to explain general trends and new phenomena will be less sensitive to sample-to-sample variation and the method used.

中文翻译：

---

几种常见的制备囊泡的方法（乳剂法除外）捕获预期的脂质比


研究人员选择不同的方法来制造巨大的单层囊泡，以满足其实验设计的不同限制。当研究人员使用各种方法时，会出现一个挑战，即即使所有实验都使用来自常见储备混合物的脂质，每种方法也可能产生具有不同平均脂质比的囊泡。在这里，我们使用质谱法研究由五种常用方法制成的囊泡溶液中的脂质比例：氧化铟锡载玻片上的电构造、铂丝上的电构造、温和的水合、乳剂转移和挤出。我们用脂质的五组分或二元混合物制成囊泡，这些脂质选择涵盖广泛的物理性质：di（18：1）PC、di（16：0）PC、di（18：1）PG、di（12：0）PE 和胆固醇。对于所有这五种脂质的混合物，ITO 电铸、Pt 电铸、温和水合和挤出方法仅导致脂质比 （≤5 mol%） 相对于普通储备液的微小变化。相比之下，乳剂转移导致储备液的胆固醇含量比预期低 ∼80%，这与所有其他磷脂相比，饱和 PC 脂质的惊人过量抵消了这一点。使用饱和和不饱和 PC 脂质和胆固醇的二元混合物的实验在很大程度上支持五组分混合物的结果。一般来说，我们的结果表明，以小步骤增加脂质比率的实验将产生对所用技术和样品间差异高度敏感的数据。例如，对于通过单一技术生产的五组分囊泡，样品间的变化为 ∼±2 mol %。 相比之下，探索脂质比的较大增量或寻求解释一般趋势和新现象的实验对样品间差异和所用方法不太敏感。