N- AND O-LINKED OLIGOSACCHARIDE PROFILING

N-和O-连接寡糖分析

• 批准号: 8363080
• 负责人: Parastoo Azadi
• 金额: $ 0.17万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363080
• 关键词: Acetic Acids; Acetone; Boric Acids; Carbohydrates; Centrifugation; Digestion; Dimethyl Sulfoxide; Excision; Funding; Glycopeptides; Grant; Hydrazine; Ice; Link; Methanol; Methods; Methylene Chloride; National Center for Research Resources; Nitrogen; Oligosaccharides; Peptides; Plant Resins; Polysaccharides; Precipitation; Preparation; Principal Investigator; Proteins; Reaction; Research; Research Infrastructure; Resources; Sampling; Sep-Pak C18; Source; Stream; Technology; Temperature; Time; Trypsin; United States National Institutes of Health; Water; acetic anhydride; base; chymotrypsin; cost; evaporation; milligram; sodium borohydride

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: Removal of contaminants by Acetone precipitation Five milligram of sample was precipitated by Acetone:Water (4:1) on ice for 30 min. The insoluble proteinaceous material was collected by centrifugation and re-precipitated three times. The final pellet of precipitated protein was dried under a stream of nitrogen. Preparation of Glycopeptides and Release of N-linked Glycans by Hydrazinolysis Two milligram of sample was digested with trypsin and chymotrypsin 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 released by Hydrazinolysis at 100 ¿C for 8 h using 200 ¿l of anhydrous hydrazine. After cooling down the sample to room temperature, hydrazine is removed under a stream of nitrogen. The sample was then re-N-acetylated using 100 ¿l of NaHCO3 and 50 ¿l of acetic anhydride on ice for 30 min. Released oligosaccharides were separated from peptide by passage through a Sep-Pak C18 cartridge column. Preparation of O-linked glycans by Reductive ¿- Elimination Two milligram of sample was subjected to alkaline reductive elimination in 100 mM NaOH containing 1.0 M sodium borohydride at 45 ¿C for 18 h. The reaction mixture was neutralized with 10% acetic acid and desalted on a column of DOWEXTM resins (50W x 8100, Sigma Aldrich). The material eluting with 5 % acetic acid was lyophilized and boric acid was removed by evaporation with methanol. Released O-glycans were purified by 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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供 该子项目的主要支持。 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源的子项目可能列出的总成本。 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 方法： 通过丙酮沉淀去除污染物 将 5 毫克样品用丙酮:水 (4:1) 在冰上沉淀 30 分钟，通过离心收集不溶性蛋白质水样物质，并重新沉淀 3 次，将沉淀的蛋白质的最终颗粒在氮气流下干燥。 。 糖肽的制备和肼解释放 N-连接聚糖 2毫克样品用胰蛋白酶和胰凝乳蛋白酶在37°C下消化18小时C 在 0.1 M Tris-HCl，pH 8.2，含有 1 mM CaCl2 中 消化产物通过 Sep-Pak C18 柱进行富集并去除污染物 富集后，糖肽在 100 ¿ C 8 小时，使用 200 ¿将样品冷却至室温后，在氮气流下除去肼，然后使用100°N重新乙酰化样品。 l NaHCO3 和 50 ¿将1μl乙酸酐置于冰上30分钟，通过Sep-Pak C18筒柱将释放的寡糖与肽分离。 通过还原法制备 O-连接聚糖 ¿ - 消除 将 2 毫克样品在含有 1.0 M 硼氢化钠的 100 mM NaOH 中于 45 °C 下进行碱性还原消除。将反应混合物用10％乙酸中和并在DOWEXTM树脂柱(50W x 8 100，Sigma Aldrich)上脱盐，将用5％乙酸洗脱的物质冻干并通过蒸发除去硼酸。释放的 O-聚糖通过 Sep-Pak C18 柱纯化。 全O-甲基化聚糖的制备 将聚糖部分溶解在二甲亚砜中，然后根据Anumula和Taylor的方法（Anumula和Taylor，1992）进行全甲基化。通过添加水来猝灭反应，并用二氯甲烷萃取全-O-甲基化碳水化合物。甲基化聚糖在氮气流下干燥。