Rapid profiling of bovine and human milk gangliosides by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry

Gangliosides are anionic glycosphingolipids widely distributed in vertebrate tissues and fluids. Their structural and quantitative expression patterns depend on phylogeny and are distinct down to the species level. In milk, gangliosides are exclusively associated with the milk fat globule membrane. They may participate in diverse biological processes but more specifically to host–pathogen interactions. However, due to the molecular complexities, the analysis needs extensive sample preparation, chromatographic separation, and even chemical reaction, which makes the process very complex and time-consuming. Here, we describe a rapid profiling method for bovine and human milk gangliosides employing matrix-assisted desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS). Prior to the analyses of biological samples, milk ganglioside standards GM3 and GD3 fractions were first analyzed in order to validate this method. High mass accuracy and high resolution obtained from MALDI FTICR MS allow for the confident assignment of chain length and degree of unsaturation of the ceramide. For the structural elucidation, tandem mass spectrometry (MS/MS), specifically as collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD) were employed. Complex ganglioside mixtures from bovine and human milk were further analyzed with this method. The samples were prepared by two consecutive chloroform/methanol extraction and solid phase extraction. We observed a number of differences between bovine milk and human milk. The common gangliosides in bovine and human milk are 2NeuAc–2Hex–Cer (GD3) and NeuAc–2Hex–Cer (GM3); whereas, the ion intensities of ganglioside species are different between two milk samples. Kendrick mass defect plot yields grouping of ganglioside peaks according to their structural similarities. Gangliosides were further probed by tandem MS to confirm the compositional and structural assignments. We found that only in human milk ganglioside was the ceramide carbon always even numbered, which is consistent with the notion that differences in the oligosaccharide and the ceramide moieties confer to their physiological distinctions.

Figure optionsDownload high-quality image (151 K)Download as PowerPoint slideResearch highlights▶ A rapid and accurate mass spectrometry-based method for profiling gangliosides was demonstrated. ▶ Ganglioside components have been accurately identified, and in order to accelerate the annotation procedure, Kendrick mass defect analysis has been applied. ▶ We obtained structural information about the polar headgroup and ceramide backbone by tandem mass spectrometry. ▶ Bovine and human milk gangliosides differ with respect to the ion intensities of the ganglioside species and the distributions of the ceramides. ▶ These results are necessary for understanding the biological structure–function relationships in complex gangliosides.