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Müller, Christoph; Junker, Julia; Bracher, Franz; Giera, Martin (2019): A gas chromatography-mass spectrometry-based whole-cell screening assay for target identification in distal cholesterol biosynthesis. In: Nature Protocols, Vol. 14, No. 8: pp. 2546-2570
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Abstract

Distal cholesterol biosynthesis (CB) has recently taken center stage as a promising drug target in several diseases previously not linked to this biochemical pathway, including cardiovascular disease, cancer, multiple sclerosis and Alzheimer's disease. Most enzymes involved in this pathway are hard to isolate, warranting dedicated analytical tools for biochemical screening. We describe the use of gas chromatography-electron ionization mass spectrometry (GC-MS) in a whole-cell screening assay aimed at monitoring interactions with all enzymes of distal CB in a single experiment. Following cell culture and lipid extraction, the trimethylsilyl ethers of sterols are analyzed by GC-MS. Analytical data for 23 relevant sterols (intermediates) are provided, allowing their unambiguous identification. Sterol pattern analysis reveals the target enzyme on the basis of characteristic marker sterols, whereas quantification of 2-C-13-acetate incorporation correlates with the inhibitory activity of drug candidates. The protocol can be used by both experienced scientists and newcomers to the field, allowing detection and quantification of small molecule-enzyme interactions in distal CB. The entire protocol can be carried out within two working days.