RELEASE OF GLYCANS, COMPOSITION ANALYSIS AND GLYCOSYL LINKAGE ANALYSIS

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, glycans were released from the glycoprotein by beta-elimination procedures as described in the previous report. Released glycans thus obtained were split into two parts, one aliquot for composition analysis and the other aliquot 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. Release of glycans from the protein Carbohydrate fractions were cleaved from the glycoprotein by [unreadable]-elimination procedures. Briefly, 500 [unreadable]L of 50 mM Sodium hydroxide (NaOH) containing 19 mg of sodium borohydride was added to the sample and incubated overnight at 45 oC. The incubated sample then was neutralized with 10% acetic acid and desalted by passing through a packed column of DOWEXTM resins (50W x 8 [unreadable]100, Sigma Aldrich) and then was lyophilized. Dried sample was cleaned of borate with methanol:acetic acid (9:1) under a stream of nitrogen gas. Then the sample was further passed through a C18 reversed phase cartridge to purify the glycans. The carbohydrate fraction was 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 dried and 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 analyzed by GC-MS. Glycosyl linkage analysis For determination of sugar linkages, partially methylated alditol acetates were prepared from permethylated glycans. Briefly, permethylated glycans were prepared based on the method of Anumula and Taylor (Anumula and Taylor, 1992) and then the sample was hydrolyzed with 2M TFA at 100 oC for 4 h, followed by reduction with NaBD4 and acetylation with acetic anhydride/pyridine (1:1, v/v) at 100 oC 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.