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: Preparation of O-glycans by Reductive ?- Elimination Five hundred microgram of PNGaseF digested glycopeptides from C33A and RP215 were 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. Permethylated O-glycans were purified by Sep-Pak C18 cartridge column. Nanospray Ionization-Linear Ion Trap Mass Spectrometry Mass analysis by NSI-MS was performed on a LTQ Orbitrap Discoverer mass spectrometer (Thermo Scientific) equipped with a nanospray ion source. Briefly, permethylated glycans were dissolved in 1 mM NaOH in 50% methanol and infused directly into a linear ion trap mass spectrometer using a nanospray source at a syringe flow rate of 0.5 ?l/min. The capillary temperature was set to 210 ?C, and MS analysis was performed in positive ion mode. Fragmentation by CID in MS/MS modes 25% collision energy was applied. The nomenclature of Domon and Costello (Domon and Costello, 1988) was used to guide the depiction of fragmentation derived from MS/MS spectra.
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