This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Preparation of the per-O-methylated carbohydratesThe carbohydrate 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. Permethylated glycans were dried under a stream of nitrogen gas.Reduction of the carboxylic acid methyl ester group on uronic acidBriefly, the permethylated samples were treated with 'Superdeuteride' (Triethylborodeuteride, LiB (C2H5)3D, Sigma-Aldrich) for 2 h at room temperature in order to reduce the carboxyl groups of uronic acid. The samples were dried and then desalted by treatment with a cation-exchange resin that was eluted with methanol/water (1:1, v/v).Glycosyl linkage analysisFor determination of sugar linkages, partially methylated alditol acectates were prepared. Briefly, permethylated glycans were hydrolysed with 2 M TFA at 100 C for 4 h, followed by reduction with 1 % NaBH4 in 30 mM 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, 20 mg/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 separations were performed on two different columns with different temperature as follows;
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