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. First of all, the sample was permethylated and an aliquot was analyzed by MALDI to confirm complete permethylation. Fully permethylated sample was then hydrolyzed, reduced and acetylated and profiled by GC-MS for the sugar linkage analysis. Detailed procedures for the linkage analysis are shown below. Preparation of the per-O-methylated carbohydrates, Cleaning up by C18 sep-pak cartridge The sample 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 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. Glycosyl linkage analysis 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. Matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF) 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. All spectra were obtained by using a 4700 Proteomics analyzer (Applied Biosystems). Gas Chromatograph-Mass Spectrometry (GC-MS) The partially methylated alditol acetates 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. 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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