Institute of Life Science, Swansea University Medical School, SA2 8PP Swansea, Wales, United Kingdom.
Advion Limited, Harlow, Essex CM20 2NQ, United Kingdom.
Proc Natl Acad Sci U S A. 2020 Mar 17;117(11):5749-5760. doi: 10.1073/pnas.1917421117. Epub 2020 Mar 4.
Dysregulated cholesterol metabolism is implicated in a number of neurological disorders. Many sterols, including cholesterol and its precursors and metabolites, are biologically active and important for proper brain function. However, spatial cholesterol metabolism in brain and the resulting sterol distributions are poorly defined. To better understand cholesterol metabolism in situ across the complex functional regions of brain, we have developed on-tissue enzyme-assisted derivatization in combination with microliquid extraction for surface analysis and liquid chromatography-mass spectrometry to locate sterols in tissue slices (10 µm) of mouse brain. The method provides sterolomic analysis at 400-µm spot diameter with a limit of quantification of 0.01 ng/mm It overcomes the limitations of previous mass spectrometry imaging techniques in analysis of low-abundance and difficult-to-ionize sterol molecules, allowing isomer differentiation and structure identification. Here we demonstrate the spatial distribution and quantification of multiple sterols involved in cholesterol metabolic pathways in wild-type and knockout mouse brain. The technology described provides a powerful tool for future studies of spatial cholesterol metabolism in healthy and diseased tissues.
胆固醇代谢失调与许多神经紊乱有关。许多固醇类物质，包括胆固醇及其前体和代谢物，具有生物活性，对大脑的正常功能非常重要。然而，大脑中的胆固醇代谢空间及其产生的固醇分布情况还没有明确的定义。为了更好地了解大脑复杂功能区的胆固醇代谢情况，我们开发了组织内酶辅助衍生化结合微液相萃取表面分析和液相色谱-质谱联用技术，以定位小鼠脑组织切片（10 µm）中的固醇类物质。该方法可在 400-µm 点直径下进行固醇组学分析，定量下限为 0.01 ng/mm，克服了之前质谱成像技术在分析低丰度和难以离子化的固醇分子方面的局限性，可实现异构体的区分和结构鉴定。本文展示了野生型和 基因敲除小鼠大脑中参与胆固醇代谢途径的多种固醇的空间分布和定量。所描述的技术为未来健康和患病组织中胆固醇代谢的空间研究提供了有力工具。
