Extended Data Fig. 4.

Mass spectrometry identification of gangliosides present in Huh-7.5 cells.

a. Extracted ion chromatograms (EIC) of GM2 ganglioside species showing their elution order.

b. Representative ESI-MS spectrum of gangliosides showing the accurate mass of [M+2H]²⁺ precursor ion of GM2 d18:1/16:0. c. Fragmentation of precursor ion (shown in b) at NCE=26 in QExactive HF. The inset shows TMT⁶ reporter ion region and the respective channel assignments. Identification of each ganglioside species is based on the presence of diagnostic B₁, O”, and Y₀’ ions. Only 14 gangliosides in Huh-7.5 cells passed these criteria (shown in d). d. Mean relative intensities ±s.e.m. of gangliosides detected by ESI-MS in 3 independent samples of Huh-7.5 cells treated with 50 or 200 μM miglustat, each with two technical replicate analyses (4 for control samples). e. Mean relative abundance ±s.e.m. of multiple GM2 ganglioside species with varying lengths of fatty acyl tail detected by ESI-MS in 3 independent samples of Huh-7.5 cells treated with miglustat at the indicated concentration. Each sample was subjected to 2 replicate ESI-MS analyses (4 for control samples). Note that the maximum concentration of miglustat used in these experiments was over 20-fold the clinical plasma Cmax of Zavesca® (miglustat) which is approximately 9 μM [European Medicines Evaluation Agency, Scientific Discussion, 2005, https://www.ema.europa.eu/en/documents/scientific-discussion/zavesca-epar-scientific-discussion_en.pdf, accessed 28 February, 2020].