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Researchers choose different methods of making giant unilamellar vesicles in order 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 5-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 5-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 roughly ±2 mol% for 5-component vesicles produced by a single technique. In contrast, experiments that explore larger lipid ratio increments or that seek to explain general trends and new phenomena will be less sensitive to sample-to-sample variation and the method used. 

Lipid peroxidation is the driver of ferroptotic cell death. However, nonconjugated and conjugated polyunsaturated fatty acids potentiate ferroptosis differently, while some isoprenoid-derived lipids inhibit ferroptosis despite being highly oxidizable. In this perspective, we propose that different oxidation mechanisms and products contribute to the discrepancies in the lipids’ potency in modulating ferroptosis. We first discuss the relative reactivities of various lipids toward two rate-determining free radical propagating mechanisms, hydrogen atom transfer (HAT) and peroxyl radical addition (PRA), and the resulting differential product profiles. We then discuss the role and regulation of lipid peroxidation in ferroptosis and the potential contributions of different oxidation products, such as truncated lipids and lipid electrophiles, from HAT and PRA mechanisms to the execution of ferroptosis. Lastly, we offer our perspective on the remaining questions to fully understand the process from lipid peroxidation to ferroptosis. 

Radical-mediated lipid oxidation and the formation of lipid hydroperoxides has been a focal point in the investigation of a number of human pathologies. Lipid peroxidation has long been linked to the inflammatory response and more recently, has been identified as the central tenet of the oxidative cell death mechanism known as ferroptosis. The formation of lipid electrophile-protein adducts has been associated with many of the disorders that involve perturbations of the cellular redox status, but the identities of adducted proteins and the effects of adduction on protein function are mostly unknown. Both cholesterol and 7-dehydrocholesterol (7-DHC), which is the immediate biosynthetic precursor to cholesterol, are oxidizable by species such as ozone and oxygen-centered free radicals. Product mixtures from radical chain processes are particularly complex, with recent studies having expanded the sets of electrophilic compounds formed. Here, we describe recent developments related to the formation of sterol-derived electrophiles and the adduction of these electrophiles to proteins. A framework for understanding sterol peroxidation mechanisms, which has significantly advanced in recent years, as well as the methods for the study of sterol electrophile-protein adduction, are presented in this review.