STRUCTURAL ANALYSIS OF N-LINKED OLIGOSACCHARIDES

N-连接低聚糖的结构分析

• 批准号: 8363063
• 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/8363063
• 关键词: Acetic Acids; Acetone; Acids; Buffers; Carbohydrates; Centrifugation; Detergents; Dimethyl Sulfoxide; Dry Ice; Enzymes; Excision; Freezing; Funding; Gases; Glycopeptides; Grant; Heating; Hour; Incubated; Ions; Isopropanol; Lasers; Link; Mass Spectrum Analysis; Methanol; Methods; Methylation; Methylene Chloride; National Center for Research Resources; Nitrogen; Oligosaccharides; Peptide N-glycohydrolase F; Peptides; Phosphate Buffer; Polysaccharides; Principal Investigator; Proteins; Reaction; Research; Research Infrastructure; Resources; Sampling; Sep-Pak C18; Series; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stream; Technology; Temperature; Time; Trypsin; Tube; United States National Institutes of Health; Vacuum; Water; cost; methyl iodide; polypeptide; 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: Purification of samples The samples were transferred into screw-cap tubes and the proteins were precipitated with cold acetone:water to remove detergents, free sugars, and other contaminants. Purification of sample was accomplished by refrigerated centrifugation and removal of supernatant. The protein pellets were dried under N2 initially and subsequently dried under vacuum. Release of N-linked glycans The samples were dissolved with 0.1 M Tris-HCl buffer (pH ~8.0) and heated at 100¿C for 5 min to denature the proteins. After cooling to room temperature, the samples were treated with trypsin and chymotypsin and incubated at 37oC overnight. The tryptic-chymotryptic digests were passed through C18 sep pak cartridges, cleaned with 5% acetic acid, and the glycopeptides/peptides were eluted subsequently in series with 20% isopropanol in 5% acetic acid, 40% isopropanol in 5% acetic acid and 100% isopropanol. The eluates was dried initially under a stream of nitrogen gas and eventually lyophilized. The dried eluates were dissolved with sodium phosphate buffer, treated with PNGase F and incubated at 37oC for 18 hours to release N-linked glycans from the polypeptide chains. After incubation, each of the enzyme (PNGase F) digests was passed through a C18 sep pak cartridge and the N-linked glycans were eluted with 5% acetic acid into screw-cap tubes. The eluates were frozen in dry ice and lyophilized. Per-O-methylation of carbohydrates The N-linked glycans were permethylated for structural characterization by mass spectrometry (Anumula and Taylor, 1992). The dried eluates were dissolved with dimethylsulfoxide and methylated with NaOH and methyl iodide. The reaction was quenched with water and per-O-methylated carbohydrates were extracted with methylene chloride and dried under N2. Profiling by Matrix-Assisted Laser-Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF MS) The permethylated glycans were dissolved with methanol and crystallized with ¿-dihyroxybenzoic acid (DHBA, 20 mg/mL in 50% methanol:water) matrix. Analysis of glycans present in the samples was performed in the positive ion mode by MALDI-TOF-MS using Bruker microflex .

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： 样品纯化 将样品转移至旋盖管中，并用冷丙酮：水沉淀蛋白质，以去除去污剂、游离糖和其他污染物。通过冷冻离心并去除上清液来完成样品的纯化。在N 2 下干燥蛋白质沉淀。最初和随后在真空下干燥。 N-连接聚糖的释放 将样品用 0.1 M Tris-HCl 缓冲液（pH ~8.0）溶解并在 100° 下加热C 5 分钟以使蛋白质变性。冷却至室温后，用胰蛋白酶和糜蛋白酶处理样品，并在 37°C 下孵育过夜。胰蛋白酶-糜蛋白酶消化物通过 C18 sep pak 柱，用 5% 乙酸清洗。随后用 20% 异丙醇的 5% 乙酸、40% 溶液串联洗脱糖肽/肽5％乙酸和100％异丙醇中的洗脱液首先在氮气流下干燥，并最终冻干。 将干燥的洗脱液用磷酸钠缓冲液溶解，用 PNGase F 处理并在 37oC 下孵育 18 小时，以从多肽链中释放 N 连接聚糖。孵育后，将每种酶 (PNGase F) 消化物通过 C18 sep。 pak 柱和 N-连接聚糖用 5% 乙酸洗脱到螺旋盖管中，将洗脱液冷冻在干冰中并冻干。 碳水化合物的全O-甲基化 将 N-连接聚糖进行全甲基化，以通过质谱分析进行结构表征（Anumula 和 Taylor，1992）。将干燥的洗脱液用二甲亚砜溶解，并用 NaOH 和碘甲烷进行甲基化。用水淬灭反应，得到全 O-甲基化碳水化合物。用二氯甲烷萃取并在N 2 下干燥。 通过基质辅助激光解吸飞行时间质谱 (MALDI-TOF MS) 进行分析 将全甲基化聚糖用甲醇溶解并用 ¿ -二羟基苯甲酸（DHBA，20 mg/mL，溶于 50% 甲醇：水）基质。使用 Bruker microflex 通过 MALDI-TOF-MS 以正离子模式对样品中存在的聚糖进行分析。