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 Glycopeptides and Release of N-linked Glycans All the six samples were freeze-dried. About 11mg of each sample was taken into 1,5 mL centrifuge tube and digested with trypsin and chymotrypsin for 22 h at 37 ?C in Ambic buffer. About 1/5 of each digestion products were enriched and freed of contaminants by Sep-Pak C18 cartridge column. After enrichment, the glycopeptides were digested with 5 ?l of PNGaseF (7.5 unit/ml) in 45 ?l of 20 mM sodium phosphate buffer, pH 7.5, for 18 h at 37 ?C. Released oligosaccharides were separated from peptide and enzyme by passage through a 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. The permethylated N-glycans were enriched and cleaned by Sep-pak C18 cartridge column. Matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF) MALDI/TOF-MS was performed in the reflector positive ion mode using ?-dihydroxybenzoic acid (DHBA, 20mg/mL solution in 50% methanol:water) as a matrix. All spectra were obtained by using a 5800 Proteomics analyzer (Applied Biosystems). NanoSpray ionization-Linear Ion Trap Mass Spectrometry (LTQ) Mass spectrometric analysis was performed following the method developed at the Complex Carbohydrates Research Center (Aoki K, Perlman M, Lim JM, Cantu R, Wells L, Tiemeyer M. J Biol Chem. 2007 Mar 23;282(12):9127-42.). Mass analysis was determined by using NSI-LTQ/MSn. Briefly, permethylated glycans were dissolved in 1mM NaOH in 50% methanol and infused directly into the instrument (LTQ,Thermo Finnigan) at a constant flow rate of 0.5 ?L/min. The capillary temperature was set at 210oC and MS analysis was performed in the positive ion mode. For total ion mapping, automated MS/MS analysis (at 35 collision energy), m/z range from 500 to 2000 was scanned in successive 2.8 mass unit windows that overlapped the preceeding window by 2 mass units
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