Several common methods of making vesicles (except an emulsion method) capture intended lipid ratios

Abstract

Researchers choose different methods of making giant unilamellar vesicles in order to satisfy different constraints of their experimental designs. A challenge of using a variety of methods is that each may produce vesicles of different lipid compositions, even if all vesicles are made from a common stock mixture. Here, we use mass spectrometry to investigate ratios of lipids in giant vesicles made by four common methods: electroformation on indium tin oxide slides, electroformation on platinum wires, gentle hydration, and emulsion transfer. The vesicles are made from a mixture of common lipid types: di(18:1)PC, di(16:0)PC, di(18:1)PG, di(12:0)PE, and cholesterol. We find that electroformation and hydration methods result in only minor shifts (≤ 5 mole%) in lipid ratio relative to the stock solution. It was previously known that the remaining method, emulsion transfer, severely reduces the amount of cholesterol in membranes. Here, we show that the emulsion transfer method also skews the ratios of lipids in a chain-dependent manner. For example, vesicles made by emulsion transfer using only PC-lipids contain less unsaturated lipid (di(16:1)PC relative to di(16:0)PC) and less shorter-chain lipid (di(16:1)PC relative to di(18:1)PC). A separate technique, extrusion, is often used by researchers to convert giant vesicles into smaller vesicles. We find that extrusion decreases cholesterol fractions (by 5.0 ± 3.3 mol% in vesicles that originally contained ∼25 mole% cholesterol). Even if all researchers were to use the same method of making vesicles, a separate question arises about whether vesicle lipid compositions vary from experiment to experiment. For the most common methods of producing GUVs (electroformation and gentle hydration), sample-to-sample variations in average lipid ratios are roughly ±2 mole%.SIGNIFICANCE STATEMENTSmall changes to the amounts and types of lipids in membranes can drastically affect the membrane’s behavior. Unfortunately, it is unknown whether (or to what extent) different methods of making vesicles alter the ratios of lipids in membranes, even when identical stock solutions are used. This presents challenges for researchers when comparing data with colleagues who use different methods. Here, we measure ratios of lipid types in vesicle membranes produced by five methods and assess each method’s reproducibility. In doing so, we provide a quantitative basis that the scientific community can use to estimate whether differences between their results can be simply attributed to differences between methods or to sample-to-sample variations.