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.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 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。 列出的子项目总成本可能 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。 研究方法： 通过丙酮沉淀去除污染物 在冰上用丙酮：水（4：1）沉淀5 mg样品30 min。离心收集不溶性蛋白质材料，再沉淀三次。在氮气流下干燥沉淀的蛋白质的最终沉淀物。 肼解法制备糖肽及N-连接聚糖的释放 在37 ℃下，在含有1 mM CaCl 2的0.1 M Tris-HCl（pH 8.2）中，用胰蛋白酶和胰凝乳蛋白酶消化2 mg样品18 h。通过Sep-Pak C18柱富集和去除污染物。富集后，使用200 μ l无水肼在100 ℃下通过肼解8 h释放糖肽。将样品冷却至室温后，在氮气流下除去联氨。然后，在冰上使用100 μ l NaHCO 3和50 μ l乙酸酐将样品重新N-乙酰化30 min。通过Sep-Pak C18筒柱将释放的寡糖与肽分离。 通过还原消除制备O-连接聚糖 在45 ℃下，在含有1.0 M硼氢化钠的100 mM NaOH中对2 mg样品进行碱性还原消除18 h。将反应混合物用10%乙酸中和，并在DOWEXTM树脂柱（50 W × 8）上脱盐100，Sigma Aldrich）。将用5%乙酸洗脱的物质冻干，用甲醇蒸发除去硼酸。通过Sep-Pak C18柱纯化释放的O-聚糖。 全-O-甲基化聚糖的制备 将聚糖组分溶于二甲亚砜中，然后根据Anumula和Taylor的方法（Anumula和Taylor，1992）进行全甲基化。通过加入水淬灭反应，用二氯甲烷萃取全-O-甲基化的碳水化合物。在氮气流下干燥全-0-甲基化聚糖。