Erwig Michelle S, Hesse Dörte, Jung Ramona B, Uecker Marina, Kusch Kathrin, Tenzer Stefan, Jahn Olaf, Werner Hauke B
Department of Neurogenetics, Max Planck Institute of Experimental Medicine, Goettingen, Germany.
Proteomics Group, Max Planck Institute of Experimental Medicine, Goettingen, Germany.
Methods Mol Biol. 2019;1936:37-63. doi: 10.1007/978-1-4939-9072-6_3.
Molecular characterization of myelin is a prerequisite for understanding the normal structure of the axon/myelin-unit in the healthy nervous system and abnormalities in myelin-related disorders. However, reliable molecular profiles necessitate very pure myelin membranes, in particular when considering the power of highly sensitive "omics"-data acquisition methods. Here, we recapitulate the history and recent applications of myelin purification. We then provide our laboratory protocols for the biochemical isolation of a highly pure myelin-enriched fraction from mouse brains and for its proteomic analysis. We also supply methodological modifications when investigating posttranslational modifications, RNA, or myelin from peripheral nerves. Notably, technical advancements in solubilizing myelin are beneficial for gel-based and gel-free myelin proteome analyses. We conclude this article by exemplifying the exceptional power of label-free proteomics in the mass-spectrometric quantification of myelin proteins.
髓磷脂的分子特征是理解健康神经系统中轴突/髓磷脂单元的正常结构以及髓磷脂相关疾病异常情况的先决条件。然而，可靠的分子图谱需要非常纯净的髓磷脂膜，尤其是在考虑高灵敏度“组学”数据采集方法的强大功能时。在这里，我们回顾了髓磷脂纯化的历史和近期应用。然后，我们提供了从鼠脑中生化分离高纯度富含髓磷脂部分及其蛋白质组分析的实验室方案。我们还提供了在研究翻译后修饰、RNA或外周神经髓磷脂时的方法改进。值得注意的是，髓磷脂溶解技术的进步有利于基于凝胶和非凝胶的髓磷脂蛋白质组分析。我们通过举例说明无标记蛋白质组学在质谱定量髓磷脂蛋白质方面的卓越能力来结束本文。
