This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Methods: Preparation of Partially Methylated Alditol Acetates (PMAAs) For determination of glycosyl linkages, PMAAs were prepared from the released permethylated N-linked glycans (remainder from oligosaccharide profiling). Briefly, permethylated glycans were hydrolyzed with HCl/water/acetic acid (0.5:1.5:8, by vol.) at 80oC for 18 h, followed by reduction with NaBD4. The latter-freed hydroxyls were acetylated with acetic anhydride/pyridine (1:1, v/v) at 100 ?C for 15 min. 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, 80 ?C (2 min) ? 180 ?C (20 ?C/min) ? 240 ?C (4 ?C/min);detector temperature, 280 ?C;inlet temperature, 250 ?C.
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