Gaucher disease (GD) is a lysosomal storage disorder that is caused by a deficiency in the lysosomal beta-glucosidase. A hallmark of GD is the massive accumulation of glucosylceramide (GlcCer) in lysosomes of tissue macrophages transforming into viable Gaucher cells. However, tissue macrophages do not manage to prevent GlcCer accumulation by conversion to GlcSph. Elucidating the chemical composition and distribution of GlcCer in primary storage cells, especially the fatty acyl composition and its glycosylation supplement, is therefore of importance to test the hypothesis that modified GlcCer is not a suitable substrate and subsequently accumulates.
Gaucher and control spleen tissue samples were either directly analyzed using DESI or total and Bligh and Dyer extracts by LC-MS on the same oa-ToF MS platform (Xevo G2-XS QTof). Frozen tissue slices were directly mounted on a modified Prosolia 2D stage and analyzed by DESI (without any additional sample pre-treatment). The LC-MS experiments were conducted using reversed phase based chromatographic methods. A typical workflow comprised of the relative quantification of the DIA LC-MS data using Progenesis QI and development informatics, providing candidate m/z target lists for the tissue lipid distribution analysis using High Definition Imaging and DriftScope software.
The multi-dimensional processed LC-MS data were submitted for untargeted searches and the detected features that demonstrated statistical relevance based on OPLS-DA were selected for compound database searching using various lipid discovery search tools. Fatty acids and glucosylceramides were found to be significantly over expressed, typically >10 fold, in GD patient samples. Despite vast morphological differences, localization/distribution and abundance differences of fatty acyls and glycosylated lipid species between the healthy control and Gaucher spleen tissue samples were readily noted. The analysis of the spleen tissue of GD patients and healthy control spleen tissue by DESI MSI and DIA LC-MS provided complementary information. The initial results suggest that the applied methodologies provide novel insights in lysosomal storage disorders, GD in particular, by revealing metabolite abnormalities, which are currently undergoing validation.