N-GLYCAN RELEASE AND GLYCOSYL LINKAGE ANALYSIS

N-聚糖释放和糖基连接分析

• 批准号: 8363091
• 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/8363091
• 关键词: Acetates; Acetic Acids; Acetonitriles; Acetylation; Acids; Aliquot; Blood capillaries; Carbohydrates; Digestion; Dimethyl Sulfoxide; Electrons; Enzymes; Funding; Gases; Glycopeptides; Grant; Ions; Lasers; Link; Mass Spectrum Analysis; Methanol; Methods; Methylene Chloride; National Center for Research Resources; Nitrogen; Oligosaccharides; Peptides; Phase; Phosphate Buffer; Polysaccharides; Preparation; Principal Investigator; Procedures; Reaction; Reporting; Research; Research Infrastructure; Resources; Sampling; Sep-Pak C18; Silicon Dioxide; Solutions; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stream; Sugar Alcohols; Technology; Temperature; Time; Trypsin; United States National Institutes of Health; Water; acetic anhydride; base; capillary; cost; detector; genetic linkage analysis; ionization; programs; pyridine; sodium phosphate; 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: First of all, N-glycans were released based on the method described in previous report. Released glycans were then permethylated and an aliquot of sample (~2%) were analyzed by MALDI for confirming fully permethylation. Fully permethylated sample was then hydrolyzed, reduced, acetylated then profiled by GC-MS. The detailed procedures used for your sample analysis are shown below. Preparation of Glycopeptides and Release of N-linked Glycans The sample was dried and digested with trypsin for 18 h at 37 ¿C in 0.1 M Tris-HCl, pH 8.2, containing 1 mM CaCl2. The digestion products were enriched and freed of contaminants by Sep-Pak C18 cartridge column. After enrichment, the glycopeptides were digested with PNGaseF (7.5 unit/ml) in 20 mM sodium phosphate buffer, pH 7.5, for 18 h at 37 ¿C. Released oligosaccharides were separated from peptide and enzyme by passage through a Sep-Pak C18 cartridge column. Preparation of the per-O-methylated glycans The glycan fraction 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 dried under a stream of nitrogen. C18 cleaning up of the permethylated N-glycans 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. 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 Microflex LRF (Bruker) 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 partially methylated alditol acetates (glycosyl linkage analysis) was performed on the on a 30m EC1 bonded phase fused silica capillary column (Alletech, Deerfield, IL) 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： 首先，根据之前报告中描述的方法释放 N-聚糖，然后将释放的聚糖进行全甲基化，并通过 MALDI 分析等份样品（~2%），以确认完全全甲基化的样品然后被水解、还原。 ，乙酰化，然后通过 GC-MS 进行分析 用于样品分析的详细程序如下所示。 糖肽的制备和 N-连接聚糖的释放 将样品干燥并用胰蛋白酶在37°C下消化18小时C 在 0.1 M Tris-HCl，pH 8.2，含有 1 mM CaCl2 中 消化产物通过 Sep-Pak C18 柱进行富集并去除污染物 富集后，用 PNGaseF（7.5 单位/ml）在 20 溶液中消化糖肽。 mM 磷酸钠缓冲液，pH 7.5，37 °C 18 小时C. 通过 Sep-Pak C18 柱将释放的寡糖与肽和酶分离。 全O-甲基化聚糖的制备 将聚糖部分溶解在二甲亚砜中，然后根据Anumula和Taylor的方法（Anumula和Taylor，1992）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。甲基化聚糖在氮气流下干燥。 全甲基化 N-聚糖的 C18 清理 简而言之，将聚糖装入 C18 sep-pak 柱中，然后用纳米纯水和 15% 乙腈洗涤。用 85% 乙腈洗脱纯化的聚糖。氮气流。 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 Microflex LRF (Bruker) 获得光谱 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 上进行组成和连接分析，在 30m EC1 上进行部分甲基化糖醇乙酸酯的分离（糖基连接分析）。键合相熔融石英毛细管柱（Alletech，Deerfield，IL），使用 80 oC 的温度程序(2 分钟) 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。