GLYCOSYL COMPOSITION AND LINKAGE OF ONE SAMPLE

一个样品的糖基组成和连接

• 批准号: 8170772
• 负责人: Parastoo Azadi
• 金额: $ 0.13万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170772
• 关键词: Acetates; Acetic Acids; Acetonitriles; Acetylation; Acids; Aliquot; Blood capillaries; Carbohydrates; Computer Retrieval of Information on Scientific Projects Database; Detergents; Dimethyl Sulfoxide; Electrons; Freeze Drying; Funding; Gases; Grant; Hexanes; Hydrolysis; Institution; Ions; Lasers; Mass Spectrum Analysis; Methanol; Methods; Methylene Chloride; Methylglycosides; Monosaccharides; Nitrogen; Phase; Polysaccharides; Preparation; Procedures; Proteomics; Reaction; Reagent; Research; Research Personnel; Resources; Sampling; Sep-Pak C18; Silicon Dioxide; Solutions; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stream; Sugar Alcohols; Temperature; Time; United States National Institutes of Health; Water; acetic anhydride; base; capillary; detector; genetic linkage analysis; ionization; programs; pyridine; sugar

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, the sample was cleaned up by C18 sep-pak column and then the sample was split into two parts, one aliquot (1/4, 32¿g) for composition analysis and the other aliquot (3/4, 96¿g) 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. C18 cleaning up of the released N-glycans The sample was passed through a C18 reversed phase cartridge for the purpose of removing possible contaminants (detergents and so on) from the carbohydrates. The carbohydrates were 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 cleaned up by hexane and dried. Then the sample was 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 extracted with dichloromethane and analyzed by GC-MS. Glycosyl linkage analysis 1) Preparation of the per-O-methylated carbohydrates The sample was permethylated for the glycosyl linkage analysis. Briefly, the sample was dissolved in dimethylsulfoxide and then permethylated based on the method of Anumula and Taylor (Anumula and Taylor, 1992) and the reaction was quenched by addition of water and per-O-methylated carbohydrates were extracted with dichloromethane. Per- O-methylated glycans were further cleaned of contaminants. Briefly, the glycans were loaded into a C18 sep-pak cartridge and then washed with nanopure water and 15% acetonitrile. The glycans were eluted with 85% acetonitrile. Purified glycans were dried under a stream of nitrogen gas. 2) Matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF) An aliquot of the permethylated sample was analyzed by MALDI to confirm complete permethylation. MALDI/TOF-MS was performed in the reflector positive ion mode using ¿-dihyroxybenzoic acid (DHBA, 20mg/mL solution in 50% methanol:water) as a matrix and the spectrum was obtained by using a 4700 Proteomics analyzer (Applied Biosystems). 3) Preparation of partially methylated alditol acectates For determination of sugar linkages, partially methylated alditol acectates were prepared from fully permethylated glycans. Briefly, permethylated glycans were hydrolysed with HCl/water/acetic acid (0.5:1.5:8, by vol.) at 80oC for 18 h, followed by reduction with 1% NaBD4 in 30mM NaOH and acetylation with acetic anhydride/pyridine (1:1, v/v) at 100 ¿C 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)160 oC (20 oC/min, 2min)200 oC (2 oC/min) 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)180 oC (20 oC/min)240 oC (4 oC/min). The detector temperature and the inlet temperature were set at 280 oC and 250 oC, respectively.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 首先，用C18 sep-pak柱净化样品，然后将样品分成两部分，一份（1/4，32¿g）用于成分分析，另一份（3/4，96¡ g) g) 用于连锁分析。按照下面所示的方法对每个等分试样进行衍生化并通过GC-MS分别进行分析。 对样品进行组成和连锁分析的详细程序如下所示。 C18 清理释放的 N-聚糖 样品通过C18反相柱，以去除碳水化合物中可能的污染物（去垢剂等）。用5%乙酸洗脱碳水化合物，并通过冻干干燥。 单糖成分分析 对于单糖组成分析，首先用新鲜制备的1M无水甲醇HCl在80℃下进行甲醇分解18小时，然后用己烷净化反应混合物。样品用甲醇/吡啶/乙酸酐（2:1:1，体积比）重新 N-乙酰化，然后用Tri-Sil试剂(Thermo Scientific)在60℃下进行三甲基硅烷化30分钟，由此获得三甲基硅烷化-甲基糖苷衍生物，用二氯甲烷萃取并通过GC-MS进行分析。 糖基连接分析 1) 全O-甲基化碳水化合物的制备 对样品进行全甲基化以进行糖基键分析，简单地说，将样品溶解在二甲基亚砜中，然后根据Anumula 和Taylor (Anumula 和Taylor, 1992)的方法进行全甲基化，并通过添加水和全-O-来猝灭反应。用二氯甲烷提取甲基化碳水化合物，进一步清除全氧甲基化聚糖中的污染物。简言之，将聚糖装入 C18 中。 sep-pak 柱，然后用纳米纯水和 15% 乙腈洗涤，用 85% 乙腈洗脱纯化的聚糖，并在氮气流下干燥。 2）基质辅助激光解吸飞行时间质谱（MALDI-TOF） 通过 MALDI 分析等份全甲基化样品，以确认完全全甲基化是使用 ¿ 在反射器正离子模式下进行的。 -二羟基苯甲酸（DHBA，20mg/mL 50%甲醇：水溶液）作为基质，使用4700蛋白质组分析仪（Applied Biosystems）获得光谱。 3) 部分甲基化糖醇乙酸酯的制备 为了测定糖键，由完全全甲基化的聚糖制备部分甲基化的糖醇乙酸酯。简言之，将全甲基化的聚糖用 HCl/水/乙酸（0.5:1.5:8，体积比）在 80℃ 水解 18 小时，然后还原。用 1% NaBD4 的 30mM NaOH 溶液并用乙酸酐/吡啶（1:1， v/v) 在 100¿通过GC-MS分析由此获得的部分甲基化的糖醇乙酸酯。 气相色谱-质谱联用仪 (GC-MS) 在连接 5970 MSD（质量选择检测器，电子轰击电离模式）的 Hewlett Packard 5890 GC 上进行组成和键合分析。在 30m EC1 键合相上进行三甲基甲硅烷基化甲基糖苷衍生物的分离（单糖组成分析）。熔融石英毛细管柱（Alletech，Deerfield，IL）在以下条件下获得。条件：烘箱温度，80℃（2分钟）160℃（20℃/分钟，2分钟）200℃（2℃/分钟）250℃（5℃/分钟）；检测器温度，310℃；入口温度，260℃；使用以下温度程序在同一毛细管柱上进行部分甲基化糖醇乙酸酯的分离（糖基连接分析） 80 oC (2 min) 180 oC (20 oC/min) 240 oC (4 oC/min) 检测器温度和入口温度分别设置为 280 oC 和 250 oC。