A monosialosyl ganglioside shown to bind specifically to uropathogenic E. coli was extracted and purified from human kidney. The complete primary structure of the ganglioside was elucidated by a combination of 1- and 2-D 1H-NMR spectroscopy, electrospray ionization mass spectroscopy (ESI-MS) and electrospray ionization/collision induced dissociation MS/MS (ESI-MS/CID-MS) of the permethylated intact compound, and methylation linkage analysis by GC/EI-MS. 1H-NMR spectroscopy at 600 MHz was carried out on 800 (g of glycosphingolipid, deuterium exchanged and dissolved in DMSO-d6/2% D2O. Following preliminary accumulation of a 1-D 1H-NMR spectrum, an assessment of glycan and ceramide structural features was made via comparison of anomeric proton and other structural reporter groups with those of known glycosphingolipids. A standard series of 2-D experiments (DQF-COSY, TOCSY, NOESY) was then performed to assign as many resonances as possible, to establish identity and anom eric form of all monosaccharide residues, and to establish sequence and as many linkage sites as possible via interglycosidic dipolar interactions. An aliquot (100 (g) was then permethylated by the Hakomori method, one half subjected to ESI-MS and -MS/MS analysis (Sciex API-III) to confirm glycan sequence and ceramide features, and the other half was depolymerized and derivatized to partially methylated alditol acetates (PMAAs) for GC-MS analysis (Hewlett-Packard 5890 GC/5970 MSD, DB-5 column). Identification of PMAAs was made by retention times and EI fragmentation patterns compared with known standards; these confirmed both the identity and linkage form of all hexose and 2-deoxy-2-acetamidohexose residues in the ganglioside. Fatty acid analysis by GC-MS of the methyl esters obtained from methanolysis of the ganglioside was used to confirm the distribution of ceramide types in this preparation. A manuscript describing this work has been submitted for publication.

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