LINKAGE ANALYSIS OF ONE SAMPLE

一个样本的连锁分析

• 批准号: 8170765
• 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/8170765
• 关键词: Acetates; Acetic Acids; Acetonitriles; Acetylation; Acids; Aliquot; Blood capillaries; Carbohydrates; Computer Retrieval of Information on Scientific Projects Database; Dimethyl Sulfoxide; Electrons; Funding; Gases; Grant; Hydrolysis; Institution; Ions; Lasers; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Methanol; Methods; Methylene Chloride; Nitrogen; Phase; Polysaccharides; Preparation; Procedures; Proteomics; Reaction; 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; 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 permethylated and an aliquot was analyzed by MALDI to confirm complete permethylation. Fully permethylated sample was then hydrolyzed, reduced and acetylated and profiled by GC-MS for the sugar linkage analysis. Detailed procedures for the linkage analysis are shown below. Preparation of the per-O-methylated carbohydrates, Cleaning up by C18 sep-pak cartridge The sample 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 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. Glycosyl linkage analysis 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. Matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF) 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. All spectra were obtained by using a 4700 Proteomics analyzer (Applied Biosystems). Gas Chromatograph-Mass Spectrometry (GC-MS) The partially methylated alditol acetates 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. Electron impact mass spectra were obtained under the following conditions: oven temperature, 80 ¿C (2 min) - 180 ¿C (20 ¿C/min) - 240 ¿C (4 ¿C/min); detector temperature, 280 ¿C; inlet temperature, 250 ¿C.

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，对样品进行全甲基化，并通过 MALDI 分析等分试样以确认完全全甲基化，然后对完全全甲基化的样品进行水解、还原和乙酰化，并通过 GC-MS 进行分析以进行糖键联分析。以下。 全氧甲基化碳水化合物的制备，通过 C18 sep-pak 柱进行净化 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。通过添加水来猝灭反应，并用二氯甲烷萃取全O-甲基化碳水化合物。简而言之，将聚糖装入 C18 sep-pak 柱中，然后用纳米纯水和 15% 洗涤。将纯化的聚糖用85％乙腈在氮气流下干燥。 糖基连接分析 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。 基质辅助激光解吸飞行时间质谱 (MALDI-TOF) MALDI/TOF-MS 在反射器正离子模式下使用 ¿ -二羟基苯甲酸（DHBA，20mg/mL 50% 甲醇：水溶液）作为基质。所有光谱均通过使用 4700 蛋白质组分析仪（Applied Biosystems）获得。 气相色谱-质谱联用仪 (GC-MS) 在连接至 5970 MSD（质量选择检测器，电子轰击电离模式）的 Hewlett Packard 5890 GC 上分析部分甲基化的糖醇乙酸酯。在 30 m EC 1 键合相熔融石英毛细管柱上进行分离。在以下条件下获得：烘箱温度，80 ¿ C（2 分钟）- 180 ¿ C (20°C/分钟) - 240°C C（4℃/分钟）；检测器温度，280℃入口温度，250°C； C.