title: Comprehensive Profiling of Glycosphingolipid Glycans Using a Novel Broad Specificity Endoglycoceramidase in a High-Throughput Workflow.
abstract: The biological function of glycosphingolipids (GSLs) is largely determined by their glycan headgroup moiety. This has placed a renewed emphasis on detailed GSL headgroup structural analysis. Comprehensive profiling of GSL headgroups in biological samples requires the use of endoglycoceramidases with broad substrate specificity and a robust workflow that enables their high-throughput analysis. We present here the first high-throughput glyco-analytical platform for GSL headgroup profiling. The workflow features enzymatic release of GSL glycans with a novel broad-specificity endoglycoceramidase I (EGCase I) from Rhodococcus triatomea, selective glycan capture on hydrazide beads on a robotics platform, 2AB-fluorescent glycan labeling, and analysis by UPLC-HILIC-FLD. R. triatomea EGCase I displayed a wider specificity than known EGCases and was able to efficiently hydrolyze gangliosides, globosides, (n)Lc-type GSLs, and cerebrosides. Our workflow was validated on purified GSL standard lipids and was applied to the characterization of GSLs extracted from several mammalian cell lines and human serum. This study should facilitate the analytical workflow in functional glycomics studies and biomarker discovery.
authors.affiliation: NIBRT GlycoScience Group, National Institute for Bioprocessing, Research and Training , Fosters Avenue, Mount Merrion, Blackrock, Dublin 4, Ireland.
authors.forename: Simone
authors.initials: S
authors.lastname: Albrecht
authors.affiliation: New England Biolabs , Ipswich, Massachusetts, United States.
authors.forename: Saulius
authors.initials: S
authors.lastname: Vainauskas
authors.affiliation: NIBRT GlycoScience Group, National Institute for Bioprocessing, Research and Training , Fosters Avenue, Mount Merrion, Blackrock, Dublin 4, Ireland.
authors.forename: Henning
authors.initials: H
authors.lastname: Stöckmann
authors.affiliation: NIBRT GlycoScience Group, National Institute for Bioprocessing, Research and Training , Fosters Avenue, Mount Merrion, Blackrock, Dublin 4, Ireland.
authors.forename: Ciara
authors.initials: C
authors.lastname: McManus
authors.affiliation: New England Biolabs , Ipswich, Massachusetts, United States.
authors.forename: Christopher H
authors.initials: CH
authors.lastname: Taron
authors.affiliation: NIBRT GlycoScience Group, National Institute for Bioprocessing, Research and Training , Fosters Avenue, Mount Merrion, Blackrock, Dublin 4, Ireland.
authors.forename: Pauline M
authors.initials: PM
authors.lastname: Rudd
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006028:Glycosphingolipids / Q000032:analysis* / Q000378:metabolism; D006367:HeLa Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D011134:Polysaccharides / Q000032:analysis* / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D006028:Glycosphingolipids; D011134:Polysaccharides; D006026:Glycoside Hydrolases; C050427:endoglycoceramidase