title: MS-based glycomic strategies for probing the structural details of polylactosaminoglycan chain on N-glycans and glycoproteomic identification of its protein carriers.
abstract: Most MS-based glycomic and glycoproteomic analyses focus on identifying changes in terminal glyco-epitopes represented by sialylation and fucosylation at specific positions of the terminal N-acetyllactosamine units. Much less attention was accorded to the underlying linear or branched poly-N-acetyllactosamine extension from the N-glycan trimannosyl core other than a simple inference of its presence due to mass data and hence glycosyl compositional assignment. Using the EA.hy926 cell line derived from human umbilical vein endothelial cells (HUVEC), we have systematically investigated the MALDI- and ESI-MS-based methodologies for probing the structural details of endothelial polylactosaminoglycans at both MS and MS(2) levels in conjunction with the use of endo-β-galactosidase to identify branching motifs and initiation sites. We showed that the polylactosaminoglycan chains on the N-glycans of EA.hy926 were less sialylated and fucosylated but more extended and branched than those of human umbilical vein endothelial cells, thus demonstrating a fundamental glycomic difference. For EA.hy926 that was investigated in more details, its polylactosaminoglycan chains were shown to be not restricted to extending from a specific antenna including the biologically important 6-arm position. Finally, experimental conditions for glycopeptide enrichment by tomato lectin were further optimized, which led to identification of over 40 candidate endothelial membrane protein carriers of polylactosaminoglycans by proteomic analysis.
authors.affiliation: Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan.
authors.forename: Shui-Hua
authors.initials: SH
authors.lastname: Wang
authors.forename: Sz-Wei
authors.initials: SW
authors.lastname: Wu
authors.forename: Kay-Hooi
authors.initials: KH
authors.lastname: Khoo
mesh_terms: D000606:Amino Sugars / Q000737:chemistry*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods; D042783:Endothelial Cells / Q000737:chemistry / Q000166:cytology; D054794:Glycomics / Q000379:methods*; D006020:Glycopeptides / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D037121:Plant Lectins / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry*; D040901:Proteomics / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
chemical_list: D000606:Amino Sugars; D006020:Glycopeptides; D006023:Glycoproteins; D037121:Plant Lectins; D011134:Polysaccharides; C039448:lactosaminoglycan; C034981:tomato lectin; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase