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.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 首先，通过C18 SEP-PAK列清理样品，然后将样品分为两个部分，一个等分试样（1/4，32¿G）进行组成分析，另一个等分试样（3/4，96¿G）进行链接分析。按照下面显示的方法衍生了每个等分试样，并分别由GC-MS分析。 在您的样本上执行的组成和链接分析的详细过程如下所示。 C18清理已释放的N-Glycans 该样品通过C18反向相墨盒，目的是从碳水合物中去除可能的污染物（洗涤剂等）。用5％的乙酸洗脱碳水合物，并通过冻干干燥。 单糖组成分析 为了单糖组成分析，制备三甲基硅烷二酰甲基二甲基糖苷衍生物。首先，用新鲜制备的1 M无水甲醇HCl在80 OC下进行18小时进行甲烷解。甲基易裂后，通过己烷清理反应混合物并干燥。然后将样品用甲基诺醇/吡啶/乙酸酐（2：1：1，vol。）重新 - 乙酰基，然后用三甲基二烯丙基二甲基二烯丙基二甲基二烯丙基二甲基二烯丙基二酰基二酰基（Tri-SIL试剂（Thermo Scientific）在60 oC中进行30分钟。用二氯甲烷提取三甲基硅烷二酰基二甲基糖苷衍生物，并通过GC-MS进行分析。 糖基链接分析 1）制备每甲基化的碳氢化物 样品被氯化以进行糖基链接分析。简而言之，将样品溶解在二甲基硫氧化氢中，然后基于Anumula和Taylor的方法（Anumula and Taylor，1992），并通过添加水和每种甲基化的碳水化的碳水化合物来淬灭反应，并用二氯甲烷提取反应。将 - 甲基化的甘氨酸进一步清洁污染物。简而言之，将聚糖加载到C18 Sep-pak弹药筒中，然后用纳米水和15％的乙腈洗涤。用85％乙腈洗脱聚糖。将纯化的聚糖干燥在氮气流下。 2）基质辅助激光解吸时间飞行时间质谱（MALDI-TOF） 通过MALDI分析了苄苄氨基化样品的等分试样，以确认完全苄苄苄化。在反射器正离子模式下使用�-二羟基苯甲酸（DHBA，20mg/ml溶液在50％甲醇：水：水）中作为基质进行MALDI/TOF -MS作为基质，并使用4700蛋白质组学分析仪（Applied Biosystems）获得光谱。 3）制备部分甲基化的醛醇加速 为了确定糖连接，从完全苄苄化聚糖中制备了部分甲基化的乙酸乙醇。简而言之，将二甲基化的聚糖用Hcl/水/乙酸水解（0.5：1.5：8，vol。）在80oC下持续18小时，然后在30mm NaOH中用1％Nabd4降低1％Nabd4，并用乙酸酸酐/吡啶乙二醇/吡啶（1：1，V/v）在100€c时以15分钟为单位。通过GC-MS分析了因此获得的部分甲基化的乙醇乙酸。 气相色谱 - 质量光谱法（GC-MS） 组成和链接分析是在连接到5970 MSD（质量选择性检测器，电子冲击电离模式）的惠普（Hewlett Packard）5890 GC上进行的。三甲基硅烷基甲基甲基糖苷衍生物（单糖组成分析）的分离在30m EC1键合融合相融合的二氧化硅毛细管柱上（Alletech，Alletech，Deerfield，IL）。在以下条件下获得了电子冲击质谱：烤箱温度，80 OC（2分钟）160 OC（20 oC/min，2 min，2 min）200 oC（2 oC/min）250 oC（5 oc/min）；检测器温度为310 OC；入口温度，260 OC。使用80 OC（2分钟）180 OC（20 oC/min）240 OC（4 oC/min）在同一毛细管柱上进行部分甲基化醛醇（糖基连接分析）的分离。检测器温度和入口温度分别设置为280 OC和250 OC。