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: First of all, N-glycans were released based on the method described in previous report. Released glycans were then permethylated and an aliquot of sample (~2%) were analyzed by MALDI for confirming fully permethylation. Fully permethylated sample was then hydrolyzed, reduced, acetylated then profiled by GC-MS. The detailed procedures used for your sample analysis are shown below. Preparation of Glycopeptides and Release of N-linked Glycans The sample was dried and digested with trypsin 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 digested with PNGaseF (7.5 unit/ml) in 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. C18 cleaning up of the permethylated N-glycans Per- O-methylated glycans were further cleaned of contaminants. Briefly, the glycans were loaded into a C18 sep-pak cartridge and then washed with nanopure water and 15% acetonitrile. The glycans were eluted with 85% acetonitrile. Purified glycans were dried under a stream of nitrogen gas. Matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF) An aliquot of the permethylated sample was analyzed by MALDI to confirm complete permethylation. MALDI/TOF-MS was performed in the reflector positive ion mode using ?-dihyroxybenzoic acid (DHBA, 20mg/mL solution in 50% methanol:water) as a matrix and the spectrum was obtained by using a Microflex LRF (Bruker) Preparation of partially methylated alditol acectates For determination of sugar linkages, partially methylated alditol acectates were prepared from fully permethylated glycans. Briefly, permethylated glycans were hydrolysed with HCl/water/acetic acid (0.5:1.5:8, by vol.) at 80oC for 18 h, followed by reduction with 1% NaBD4 in 30mM NaOH and acetylation with acetic anhydride/pyridine (1:1, v/v) at 100 ?C for 15 min. The partially methylated alditol acetates thus obtained were analyzed by GC-MS. Gas Chromatograph-Mass Spectrometry (GC-MS) The composition and linkage analysis were performed on a Hewlett Packard 5890 GC interfaced to a 5970 MSD (mass selective detector, electron impact ionization mode). The separation of the partially methylated alditol acetates (glycosyl linkage analysis) was performed on the on a 30m EC1 bonded phase fused silica capillary column (Alletech, Deerfield, IL) using a temperature program of 80 oC (2 min)?180 oC (20 oC/min)?240 oC (4 oC/min). The detector temperature and the inlet temperature were set at 280 oC and 250 oC, respectively.
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