Analytical Methods

Sterols and oxysterols were analysed by LC-ESI-MSⁿ using a charge-tagging approach (enzyme-assisted derivatisation for sterol analysis, EADSA) described fully in [32,33] and in supplementary information. In brief, non-polar sterols including cholesterol, 7-DHC and 8-DHC were separated from more-polar oxysterols by reversed-phase solid phase extraction (RP-SPE). The separated fractions were individually treated with cholesterol oxidase to convert 3β-hydroxy-5-ene and 3β-hydroxy-5,7(or 8)-diene to their 3-oxo-4-ene and 3-oxo-4,7(or 8)–diene equivalents, then derivatised with Girard P (GP) reagent (Supplementary Figure S1A) to add a charged quaternary nitrogen group to the analytes which greatly improve their LC-ESI-MS and MSⁿ response. When fragmented by MS² GP-tagged analytes give an intense [M-Py]⁺ ion, corresponding to the loss of the pyridine (Py) ring (Figure S1B), which can be fragmented further by MS³ to give a structurally informative pattern (Figure S1C–J). Some sterols and oxysterols naturally contain an oxo group and can be differentiated from those oxidised to contain one by omitting the cholesterol oxidase enzyme from the sample work-up procedure [32]. No hydrolysis step was carried out so free sterols and oxysterols were exclusively analysed.