A simple and accurate micromethod for quantitative determination of ganglioside patterns

Gangliosides are an important class of glycosphingolipids involved in numerous biological processes such as neuronal development, host–pathogen interactions and gastrointestinal health. Due to the highly heterogeneous nature and relatively low abundance of gangliosides, characterization of gangliosides in biological membranes is challenging. Existing methods for ganglioside analysis are quite time consuming and require expensive high resolution mass spectrometers. A rapid method combining reversed phase chromatography and mass spectrometry was developed using a triple-quadrupole mass spectrometer operating in multiple reaction monitoring mode. The ganglioside species were separated with a Poroshell 120 EC-C18 column and analysed under the negative ion mode. This method allows a sensitive, specific, and quantitative assay for profiling gangliosides. The method is developed for analysis of gangliosides in the milk fat globule membrane of whole milk and applied to other biological membranes. Application includes the cellular membrane of prostate cancer cells. In summary, the method allows various biological membranes to be screened for over 600 gangliosides from 12 classes (GM1, GM2, GM3, GM4, GD1, GD2, GD3, GD4, GT1, GT2, GT3, and GT4) in less than three hours. In summary, expressed as % of relative amounts: 1.5% GM3, 80.2% GD3, 14.4% GT3, 1.5% GM1, 2.4% GD1 were observed in whole milk; 2.5% GD1, 88.2% GD3, 2.5% GM1, 2.2% GM3, 0.2% GT2, 4.2% GT3 were observed in buttermilk and 10.6% GD1, 55.6% GD3, 1.6% GM1, 12.2% GM3, 19.2% GT3, 0.9% GT4 were observed in colostrum.