LINKED GLYCOSYLS ANALYSIS BY GC-MS

通过 GC-MS 进行连接糖基分析

• 批准号: 8363065
• 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/8363065
• 关键词: Acetates; Blood capillaries; Code; Electrons; Funding; Gases; Grant; Hydrolysis; Hydroxyl Radical; Link; Mass Spectrum Analysis; Methods; Methylene Chloride; National Center for Research Resources; Phase; Polysaccharides; Preparation; Principal Investigator; Research; Research Infrastructure; Resources; Silicon Dioxide; Source; Sugar Alcohols; Technology; Temperature; Trifluoroacetic Acid; United States National Institutes of Health; acetic anhydride; capillary; cost; detector; ionization; pyridine

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: Linked Glycosyl Composition Analysis Preparation of Partially Methylated Alditol Acetates (PMAAs) Partially methylated alditol acetates were prepared from the remainder of permethylated O-linked glycans of CA215 Lot # S15K, s100425 (refer to CCRC code # GL042810Z). Briefly, permethylated glycans were hydrolyzed with 2N trifluoroacetic acid at 121oC for 2 hr, followed by reduction with NaBD4. After hydrolysis and reduction steps, the free hydroxyls of the partially methylated alditols were acetylated with acetic anhydride:pyridine (1:1, v/v) at 100oC for 1 hr to produce PMAAs. Partially methylated alditol acetates were extracted with methylene chloride. Gas Chromatograph-Mass Spectrometry (GC-MS) The PMAAs were analyzed on a Hewlett Packard 5890 GC interfaced to a 5970 MSD (mass selective detector, electron impact ionization mode). The separation was performed on a 30 m EC 1 bonded phase fused silica capillary column (Altech). Electron impact mass spectra were obtained under the following conditions: oven temperature, 140¿C (2.0¿C/min) ¿ 220¿C (20¿C/min) ¿ 300¿C (7.5 min); detector temperature, 280 ¿C; inlet temperature, 250 ¿C.

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： 连接糖基组成分析 部分甲基化糖醇乙酸酯 (PMAA) 的制备 部分甲基化的糖醇乙酸酯由 CA215 批次 # S15K、s100425 的剩余全甲基化 O 连接聚糖制备（参见 CCRC 代码 # GL042810Z）。简而言之，将全甲基化聚糖用 2N 三氟乙酸在 121℃ 下水解 2 小时，然后还原。在水解和还原步骤之后，部分甲基化的糖醇的游离羟基用乙酸酐:吡啶(1:1，v/v)在100℃下乙酰化1小时以产生PMAA，用二氯甲烷萃取部分甲基化的糖醇乙酸酯。 气相色谱-质谱联用仪 (GC-MS) PMAA 在与 5970 MSD 连接的 Hewlett Packard 5890 GC 上进行分析。分离在 30 m EC 1 键合相熔融石英毛细管柱 (Altech) 上进行。在以下条件下获得：烤箱温度，140° C (2.0°C/分钟) ¿ 220° C (20°C/分钟) ¿ 300€ C（7.5 分钟）；检测器温度，280 ¿入口温度，250°C； C.