Lipid rafts, membrane microdomains enriched with (glyco)sphingolipids, cholesterol, and select proteins, act as cellular signalosomes. Various methods have been used to separate lipid rafts from bulk (non-raft) membranes, but most often, non-ionic detergent Triton X-100 has been used in their isolation. However, Triton X-100 is a reported disruptor of lipid rafts. Histological evidence confirmed raft disruption by Triton X-100, but remarkably revealed raft stability to treatment with a related polyethylene oxide detergent, Brij O20. We report isolation of detergent-resistant membranes from mouse brain using Brij O20 and its use to determine the distribution of major mammalian brain gangliosides, GM1, GD1a, GD1b and GT1b. A different distribution of gangliosides—classically used as a raft marker—was discovered using Brij O20 versus Triton X-100. Immunohistochemistry and imaging mass spectrometry confirm the results. Use of Brij O20 results in a distinctive membrane distribution of gangliosides that is not all lipid raft associated, but depends on the ganglioside structure. This is the first report of a significant proportion of gangliosides outside raft domains. We also determined the distribution of proteins functionally related to neuroplasticity and known to be affected by ganglioside environment, glutamate receptor subunit 2, amyloid precursor protein and neuroplastin and report the lipid raft populations of these proteins in mouse brain tissue. This work will enable more accurate lipid raft analysis with respect to glycosphingolipid and membrane protein composition and lead to improved resolution of lipid–protein interactions within biological membranes.

中文翻译：

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谁进来，谁出去？重新评估脂筏居民

脂筏，富含（糖）鞘脂、胆固醇和选择蛋白质的膜微结构域，充当细胞信号体。已使用各种方法将脂筏与散装（非筏）膜分离，但大多数情况下，非离子洗涤剂 Triton X-100 已用于分离它们。然而，据报道，Triton X-100 是脂筏的破坏者。组织学证据证实了 Triton X-100 对筏板的破坏，但显着地揭示了用相关的聚环氧乙烷洗涤剂 Brij O20 处理的筏板稳定性。我们报告了使用 Brij O20 从小鼠脑中分离出耐洗涤剂膜及其用于确定主要哺乳动物脑神经节苷脂、GM1、GD1a、GD1b 和 GT1b 的分布。使用 Brij O20 与 Triton X-100 发现了神经节苷脂的不同分布——通常用作筏标记物。免疫组织化学和成像质谱证实了结果。Brij O20 的使用导致神经节苷脂的独特膜分布，这并非所有脂筏相关，而是取决于神经节苷脂结构。这是在筏域外有很大比例的神经节苷脂的第一份报告。我们还确定了与神经可塑性功能相关的蛋白质的分布，这些蛋白质已知受神经节苷脂环境、谷氨酸受体亚基 2、淀粉样蛋白前体蛋白和神经塑性蛋白的影响，并报告了这些蛋白质在小鼠脑组织中的脂筏种群。这项工作将能够对鞘糖脂和膜蛋白组成进行更准确的脂筏分析，并提高生物膜内脂蛋白相互作用的分辨率。