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. Dialysis The samples were dialyzed using a Tube-O-Dialyzer (4.0 kDa cut-off membrane;G BioSciences) against nanopure water at 4oC for about 22 hours to remove salts and other contaminants. Nanopure water was replaced three times during the entire dialysis period. Release of O-linked glycans by ?-elimination O-linked carbohydrates were cleaved from the samples by ?-elimination procedure. Briefly, 250 ?L of 50 mM NaOH was added to each of the samples and then checked for pH. Upon determination that the pH was basic, another 250 ?L of 50 mM NaOH containing 19 mg of sodium borohydride was added to each of the samples, vortexed, and incubated overnight at 450C. The incubated samples then were neutralized with 10% acetic acid and desalted by passing through a packed column of Dowex resins and then were lyophilized. Dried samples were cleaned of borate with methanol:acetic acid (9:1) under a stream of nitrogen gas before permethylation. Per-O-methylation of O-linked glycans Released O-linked glycans were permethylated and were evaluated by mass spectrometry (Anumula and Taylor, 1992) to verify if the derivatization was carried to completion. The glycans were dissolved with dimethylsulfoxide and then methylated with NaOH and methyl iodide. The reaction was quenched with water and per-O-methylated carbohydrates were extracted with methylene chloride. Glycosyl Linkage Analysis For determination of glycosyl linkages, partially methylated alditol acetates (PMAAs) were prepared from the released permethylated O-linked glycans. Briefly, permethylated glycans were hydrolyzed with 2N trifluoroacetic acid (TFA) at 100oC for 4 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 Chromatography-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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