LINKED GLYCOSYLS ANALYSIS BY GC-MS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Methods: Linked Glycosyl Composition Analysis Preparation of Partially Methylated Alditol Acetates (PMAAs) Partially methylated alditol acetates were prepared from the remainder of permethylated O-linked glycans of CA215 Lot # S15K, s100425 (refer to CCRC code # GL042810Z). Briefly, permethylated glycans were hydrolyzed with 2N trifluoroacetic acid at 121oC for 2 hr, followed by reduction with NaBD4. After hydrolysis and reduction steps, the free hydroxyls of the partially methylated alditols were acetylated with acetic anhydride:pyridine (1:1, v/v) at 100oC for 1 hr to produce PMAAs. Partially methylated alditol acetates were extracted with methylene chloride. Gas Chromatograph-Mass Spectrometry (GC-MS) The PMAAs were analyzed on a Hewlett Packard 5890 GC interfaced to a 5970 MSD (mass selective detector, electron impact ionization mode). The separation was performed on a 30 m EC 1 bonded phase fused silica capillary column (Altech). Electron impact mass spectra were obtained under the following conditions: oven temperature, 140¿C (2.0¿C/min) ¿ 220¿C (20¿C/min) ¿ 300¿C (7.5 min); detector temperature, 280 ¿C; inlet temperature, 250 ¿C.