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: Removal of contaminants by Acetone precipitation Five milligram of sample was precipitated by Acetone:Water (4:1) on ice for 30 min. The insoluble proteinaceous material was collected by centrifugation and re-precipitated three times. The final pellet of precipitated protein was dried under a stream of nitrogen. Preparation of Glycopeptides and Release of N-linked Glycans by Hydrazinolysis Two milligram of sample was digested with trypsin and chymotrypsin for 18 h at 37 ?C in 0.1 M Tris-HCl, pH 8.2, containing 1 mM CaCl2. The digestion products were enriched and freed of contaminants by Sep-Pak C18 cartridge column. After enrichment, the glycopeptides were released by Hydrazinolysis at 100 ?C for 8 h using 200 ?l of anhydrous hydrazine. After cooling down the sample to room temperature, hydrazine is removed under a stream of nitrogen. The sample was then re-N-acetylated using 100 ?l of NaHCO3 and 50 ?l of acetic anhydride on ice for 30 min. Released oligosaccharides were separated from peptide by passage through a Sep-Pak C18 cartridge column. Preparation of O-linked glycans by Reductive ?- Elimination Two milligram of sample was subjected to alkaline reductive elimination in 100 mM NaOH containing 1.0 M sodium borohydride at 45 ?C for 18 h. The reaction mixture was neutralized with 10% acetic acid and desalted on a column of DOWEXTM resins (50W x 8?100, Sigma Aldrich). The material eluting with 5 % acetic acid was lyophilized and boric acid was removed by evaporation with methanol. Released O-glycans were purified by Sep-Pak C18 cartridge column. Preparation of the per-O-methylated glycans The glycan fraction was dissolved in dimethylsulfoxide and then permethylated based on the method of Anumula and Taylor (Anumula and Taylor, 1992). The reaction was quenched by addition of water and per-O-methylated carbohydrates were extracted with dichloromethane. Per- O-methylated glycans were dried under a stream of nitrogen.
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