LINKED GLYCOSYL ANALYSIS OF GLYCOPROTEIN N-LINKED OLIGOSACCHARIDES

糖蛋白 N-连接寡糖的连接糖基分析

• 批准号: 8363086
• 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/8363086
• 关键词: Acetates; Acetic Acids; Amino Sugars; Blood capillaries; Funding; Gases; Glycoproteins; Grant; Hydrolysis; Hydroxyl Radical; Link; Mass Spectrum Analysis; Methods; National Center for Research Resources; Oligosaccharides; Phase; Polysaccharides; Preparation; Principal Investigator; Ramp; Research; Research Infrastructure; Resources; Silicon Dioxide; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sugar Alcohols; Technology; Temperature; United States National Institutes of Health; Water; acetic anhydride; capillary; cost; pyridine; sugar

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: Preparation of Partially Methylated Alditol Acetates (PMAAs) For the determination of linked glycosyls of that composed the N-linked glycans, PMAAs were prepared from the permethylated oligosaccharides (remainder from oligosaccharide profiling by MALDI-TOF/TOF MS). Briefly, permethylated oligosaccharides were hydrolyzed with HCL:water:acetic acid (1:3:16) at 80oC overnight, followed by reduction with NaBD4. The latter-freed hydroxyls after hydrolysis were acetylated with acetic anhydride/pyridine (1:1, v/v) at 100 ¿C for 1 h. Gas Chromatograph-Mass Spectrometry (GC-MS) The PMAAs for amino sugars were analyzed on a Hewlett Packard 5890 GC interfaced to a 5970 MSD, which was installed with a 30-m EC 1 bonded phase fused silica capillary column (Altech), whereas the neutral sugars were analyzed on an Agilent 7890A GC interfaced to a 5975C MSD equipped with a Rtx-2330 column. The GC conditions were the following: For amino sugars ¿ initial oven temperature was 140¿C (2.0¿C/min), which was held for 0.5 min, ramped to 220¿C (20¿C/min) and finally ramped to 300¿C (20¿C/min) and held for 7.5 min. For neutral sugars ¿ initial oven temp was 80oC, which was held for 2 min, ramped to 170oC at 30oC/min, held for 0 min, and finally ramped to 235oC at 4oC/min and held for 20 min

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： 部分甲基化糖醇乙酸酯 (PMAA) 的制备 为了确定组成 N-连接聚糖的连接糖基，从全甲基化寡糖（通过 MALDI-TOF/TOF MS 进行寡糖分析的剩余部分）制备 PMAA。简而言之，用 HCL:水:乙酸水解全甲基化寡糖（ 1:3:16) 在 80oC 过夜，然后用 NaBD4 还原。水解后的羟基用乙酸酐/吡啶（1:1，v/v）在100℃下乙酰化C 1小时。 气相色谱-质谱联用仪 (GC-MS) 氨基糖的 PMAA 在连接至 5970 MSD 的 Hewlett Packard 5890 GC 上进行分析，5970 MSD 安装有 30 米 EC 1 键合相熔融石英毛细管柱 (Altech)，而中性糖则在 Agilent 7890A GC 上进行分析连接至配备 Rtx-2330 色谱柱的 5975C MSD GC 条件为以下：对于氨基糖 ¿烤箱初始温度为 140°C C (2.0°C/min)，保持 0.5 分钟，升至 220°C C（20°C/分钟），最后升至 300°C C (20°C/分钟) 并保持 7.5 分钟 对于中性糖 ¿初始烘箱温度为80℃，保持2分钟，以30℃/分钟升温至170℃，保持0分钟，最后以4℃/分钟升温至235℃，保持20分钟