Structural characterization and analysis of different epimers of neutral glycosphingolipid LcGg4 by ion mobility spectrometry-mass spectrometry.

LcGg4, a neutral glycosphingolipid (GSL) and cancer antigen, its epimers GalNAc-LcGg4 and GlcNAc-LcGg4, and three lipid forms of GalNAc-LcGg4 were studied by mass spectrometry (MS). It was found that different forms of GalNAc-LcGg4 carrying homologous (d16:1/18:0) and (d18:1/18:0) lipids were easily separated and identified using liquid chromatography (LC)-MS. In addition, like gangliosides, homologous lipid forms of GalNAc-LcGg4 showed the same fragmentation pattern, except for a uniform shift of their glycolipid product ions by a certain m/z number determined by the varied lipid structure. It was also disclosed that LcGg4 and its epimers GalNAc-LcGg4 and GlcNAc-LcGg4, which are different only in the C4-configuration of their non-reducing end sugar residues, gave the same MS/MS product ions in similar relative intensities, as well as the same LC retention time, suggesting the challenge to differentiate epimeric GSLs by LC-MS. However, ion mobility spectrometry (IMS)-MS was able to efficiently separate and distinguish these epimers. This study has demonstrated the promise of IMS-MS for isomeric GSL characterization and the IMS-MS and LC-MS/MS combination for natural GSL analysis.

Analysis and Comparison of Mouse and Human Brain Gangliosides via Two-Stage Matching of MS/MS Spectra.

Glycosphingolipids (GSLs), including gangliosides, are essential components of the cell membrane. Because of their vital biological functions, a facile method for the analysis and comparison of GSLs in biological issues is desired. To this end, a new method for GSL analysis was developed based on two-stage matching of the carbohydrate and glycolipid product ions of experimental and reference MS/MS spectra of GSLs. The applicability of this method to the analysis of gangliosides in biological tissues was verified using human plasma and mouse brains spiked with standards. The method was then used to characterize endogenous gangliosides in mouse and human brains. It was shown that each endogenous ganglioside species had varied lipid forms and that mouse and human brains had different compositions of ganglioside species and lipid forms. Moreover, a 36-carbon ceramide is found to represent the major lipid form for mouse brain gangliosides, while the major lipid form for most human brain gangliosides is a 38-carbon ceramide. This study has verified that the two-stage MS/MS spectral matching method could be used to study gangliosides or GSLs and their lipid forms in complex biological samples, thereby having a broad application.

Characterization of Glycosphingolipids and Their Diverse Lipid Forms through Two-Stage Matching of LC-MS/MS Spectra.

Glycosphingolipids (GSLs) play a key role in various biological and pathological events. Thus, determination of the complete GSL compositions in human tissues is essential for comparative and functional studies of GSLs. In this work, a new strategy was developed for GSL characterization and glycolipidomics analysis based on two-stage matching of experimental and reference MS/MS spectra. In the first stage, carbohydrate fragments, which contain only glycans and thus are conserved within a GSL species, are directly matched to yield a species identification. In the second stage, glycolipid fragments from the matched GSL species, which contain both the lipid and glycans and thus shift due to lipid structural changes, are treated according to lipid rule-based matching to characterize the lipid compositions. This new strategy uses the whole spectrum for GSL characterization. Furthermore, simple databases containing only a single lipid form per GSL species can be utilized to identify multiple GSL lipid forms. It is expected that this method will help accelerate glycolipidomics analysis and disclose new and diverse lipid forms of GSLs.