GLYCOSYL COMPOSITION AND LINKAGE OF ONE SAMPLE

University Of Georgia, Athens GA

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Abstract

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 cleaned up by C18 sep-pak column and then the sample was split into two parts, one aliquot (1/4, 32[unreadable]g) for composition analysis and the other aliquot (3/4, 96[unreadable]g) for linkage analysis. Each aliquot was derivatized following the methods shown below and analyzed by GC-MS separately. The detailed procedures for composition and linkage analyses performed on your sample are shown below. C18 cleaning up of the released N-glycans The sample was passed through a C18 reversed phase cartridge for the purpose of removing possible contaminants (detergents and so on) from the carbohydrates. The carbohydrates were eluted with 5% acetic acid and dried by lyophilization. Monosaccharide composition analysis For monosaccharide composition analysis, trimethylsilylated-methylglycoside derivatives were prepared. First of all, methanolysis was performed with freshly prepared 1 M anhydrous methanolic HCl at 80 oC for 18 h. After methanolysis, the reaction mixture was cleaned up by hexane and dried. Then the sample was re-N-acetylated with methanol/pyridine/acetic anhydride (2:1:1, by vol.) followed by trimethylsilylation with Tri-Sil reagent (Thermo scientific) at 60 oC for 30 min. The trimethylsilylated-methylglycoside derivatives thus obtained were extracted with dichloromethane and analyzed by GC-MS. Glycosyl linkage analysis 1) Preparation of the per-O-methylated carbohydrates The sample was permethylated for the glycosyl linkage analysis. Briefly, the sample was dissolved in dimethylsulfoxide and then permethylated based on the method of Anumula and Taylor (Anumula and Taylor, 1992) and 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. 2) 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 [unreadable]-dihyroxybenzoic acid (DHBA, 20mg/mL solution in 50% methanol:water) as a matrix and the spectrum was obtained by using a 4700 Proteomics analyzer (Applied Biosystems). 3) 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 [unreadable]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 trimethylsilylated-methylglycoside derivatives (monosaccharide composition analysis) was performed on a 30m EC1 bonded phase fused silica capillary column (Alletech, Deerfield, IL). Electron impact mass spectra were obtained under the following conditions: oven temperature, 80 oC (2 min)[unreadable]160 oC (20 oC/min, 2min)[unreadable]200 oC (2 oC/min) [unreadable]250 oC (5 oC/min);detector temperature, 310 oC;inlet temperature, 260 oC. The separation of the partially methylated alditol acetates (glycosyl linkage analysis) was performed on the same capillary column using a temperature program of 80 oC (2 min)[unreadable]180 oC (20 oC/min)[unreadable]240 oC (4 oC/min). The detector temperature and the inlet temperature were set at 280 oC and 250 oC, respectively.