STRUCTURAL ANALYSIS OF O-LINKED OLIGOSACCHARIDES

O-连接低聚糖的结构分析

• 批准号: 8363055
• 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/8363055
• 关键词: Acetic Acids; Acids; Borates; Buffers; Carbohydrates; Cleaved cell; Dimethyl Sulfoxide; Enzymes; 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; Plant Resins; Polysaccharides; Principal Investigator; Procedures; 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; Water; cost; methyl iodide; polypeptide; sodium borohydride; sodium phosphate

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: Release of N-linked glycans The samples were transferred into microcentrifuge tubes, lyophilized, dissolved with 0.1 M Tris-HCl buffer (pH ~8.0) and heated at 100¿C for 5 min to denature the protein. After cooling to room temperature, the samples were treated with trypsin and chymotypsin and incubated at 37oC overnight. Each of the tryptic-chymotryptic digests was passed through a C18 sep pak cartridge, cleaned with 5% acetic acid, and 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 were 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, the enzyme (PNGase F) digests were passed through C18 sep pak cartridges and the N-linked glycans of each sample were eluted first into a tube with 5% acetic acid followed by the elution of O-linked glycopeptides/peptides into another tube with 20% isopropanol in 5% acetic acid, 40% isopropanol in 5% acetic acid and 100% isopropanol. The O-linked glycopeptides/peptides fraction was dried initially under a stream of N2 to evaporate the isopropanol and lyophilized eventually. Release of O-linked glycans by ¿-elimination O-linked carbohydrates were cleaved from the glycopeptides by ¿-elimination procedure. Briefly, 250 ¿L of 50 mM NaOH were added to each of the samples and then checked for pH. Upon determination that the pH was basic, another 250 ¿L of 50 mM NaOH containing 19 mg of sodium borohydride were added to the samples, vortexed, and incubated overnight at 450C. The incubated samples then were neutralized with 10% acetic acid and desalted by passing through a packed column of Dowex resins and then were lyophilized. Dried samples were cleaned of borate with methanol:acetic acid (9:1) under a stream of nitrogen gas before permethylation. Per-O-methylation of carbohydrates The O-linked glycans from both samples 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： N-连接聚糖的释放 将样品转移至微量离心管中，冻干，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。每种胰蛋白酶-胰凝乳蛋白酶消化物均通过用 5% 乙酸清洗的 C18 sep pak 柱。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 37°C 下孵育 18 小时，以从多肽链中释放 N 连接聚糖。孵育后，将酶 (PNGase F) 消化物通过 C18 sep pak 柱并进行纯化。首先将每个样品的 N-连接聚糖洗脱到装有 5% 乙酸的试管中，然后将 O-连接糖肽/肽洗脱到另一管装有 5% 乙酸中的 20% 异丙醇、5% 乙酸中的 40% 异丙醇和 100% 异丙醇。 O-连接糖肽/肽级分最初在 N2 流下干燥，以蒸发异丙醇并最终冻干。 通过 ¿ 释放 O-连接聚糖-消除 O-连接碳水化合物通过 ¿ 从糖肽上裂解下来- 简单地说，250 ¿将 L 50 mM NaOH 添加到每个样品中，然后检查 pH 值，确定 pH 值呈碱性后，再检查 250 ¿将含有 19 mg 硼氢化钠的 L 50 mM NaOH 添加到样品中，涡旋并在 450°C 下孵育过夜。然后用 10% 乙酸中和孵育的样品，并通过 Dowex 树脂填充柱脱盐，然后进行脱盐。在全甲基化之前，在氮气流下用甲醇:乙酸 (9:1) 清除干燥样品中的硼酸盐。 碳水化合物的全O-甲基化 将两个样品中的 O-连接聚糖进行全甲基化，以通过质谱进行结构表征（Anumula 和 Taylor，1992）。将干燥的洗脱液用二甲亚砜溶解，并用 NaOH 和甲基碘进行甲基化。用水和全 O- 猝灭反应。用二氯甲烷提取甲基化碳水化合物并在氮气下干燥。 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。