A platform for the structural characterization of glycans enzymatically released from glycosphingolipids extracted from tissue and cells
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Rationale: Glycosphingolipids (GSLs) constitute a highly diverse class of glyco-conjugates which are involved in many aspects of cell membrane function and disease. The isolation, detection and structural characterization of the carbohydrate (glycan) component of GSLs are particularly challenging given their structural heterogeneity and thus rely on the development of sensitive, analytical technologies. Methods: Neutral and acidic GSL standards were immobilized onto polyvinylidene difluoride (PVDF) membranes and glycans were enzymatically released using endoglycoceramidase II (EGCase II), separated by porous graphitized carbon (PGC) liquid chromatography and structurally characterized by negative ion mode electrospray ionization tandem mass spectrometry (PGC-LC/ESI-MS/MS). This approach was then employed for GSLs isolated from 100 mg of serous and endometrioid cancer tissue and from cell line (107 cells) samples. Results: Glycans were released from GSL standards comprising of ganglio-, asialo-ganglio- and the relatively resistant globo-series glycans, using as little as 1 mU of enzyme and 2 µg of GSL. The platform of analysis was then applied to GSLs isolated from tissue and cell line samples and the released isomeric and isobaric glycan structures were chromatographically resolved on PGC and characterized by comparison with the MS2 fragment ion spectra of the glycan standards and by application of known structural MS2 fragment ions. This approach identified several (neo-)lacto-, globo- and ganglio-series glycans and facilitated the discrimination of isomeric structures containing Lewis A, H type 1 and type 2 blood group antigens and sialyl-tetraosylceramides. Conclusion: We describe a relatively simple, detergent-free, enzymatic release of glycans from PVDF-immobilized GSLs, followed by the detailed structural analysis afforded by PGC-LC-ESI-MS/MS, to offer a versatile method for the analysis of tumour and cell-derived GSL-glycans. The method uses the potential of MS2 fragmentation in negative ion ESI mode to characterize, in detail, the biologically relevant glycan structures derived from GSLs. Copyright © 2015 John Wiley & Sons, Ltd.
Rapid Communications in Mass Spectrometry
Biochemistry and Cell Biology not elsewhere classified