O-LINKED OLIGOSACCHARIDE PROFILING AND LINKAGE ANALYSIS OF ONE SAMPLE

一个样品的 O-连接寡糖图谱和连锁分析

• 批准号: 7722692
• 负责人: Parastoo Azadi
• 金额: $ 0.02万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-08 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7722692
• 关键词: 1-Propanol; Acetates; Acetic Acid; Acetic Acids; Acetonitriles; Acetylation; Acids; Borates; Carbohydrates; Cleaved cell; Computer Retrieval of Information on Scientific Projects Database; Dimethyl Sulfoxide; Electrons; Excision; Freeze Drying; Funding; Gases; Glycopeptides; Grant; Hydrolysis; Incubated; Institution; Ions; Lasers; Link; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Methanol; Methods; Methylene Chloride; Nitrogen; Oligosaccharides; Peptides; Phase; Plant Resins; Polysaccharides; Preparation; Procedures; Propanols; Proteomics; Reaction; Research; Research Personnel; Resources; Sampling; Sep-Pak C18; Series; Solutions; Source; Speed; Stream; Sugar Alcohols; Temperature; Time; Tube; United States National Institutes of Health; Vacuum; Water; acetic anhydride; base; detector; genetic linkage analysis; ionization; pyridine; sodium borohydride; 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. ¿- elimination, Desalting, Borate removal, Cleaning by C18 sep-pak cartridge O-linked carbohydrate fractions were cleaved from the glycopeptides by ¿-elimination procedures. Briefly, 1M sodium borohydride (NaBH4) in 50 mM Sodiumhydroxide (NaOH) was added to the sample and incubated 17 h at 45oC. The incubated sample then was neutralized with 10% acetic acid and desalted by passing through a packed column of DOWEXTM resins (50W x 8  100, Sigma Aldrich) and then was lyophilized. Dried sample was cleaned of borate with methanol:acetic acid (9:1, v/v) under a stream of nitrogen gas. Then the sample was passed through a C18 reversed phase cartridge to purify the O-glycans. The carbohydrate fractions (O-linked glycans) were first eluted with 5% acetic acid and then the peptides were eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and 100% iso-propanol each into a microcentrifuge tube. The carbohydrate fraction was dried by lyophilization, whereas the peptide fractions were dried in the speed vacuum concentrator and then combined in one tube. Preparation of the per-O-methylated carbohydrates, Cleaning up by C18 sep-pak cartridge The carbohydrate 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 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 then 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 permethylated O-glycans. Briefly, permethylated glycans were hydrolysed with 2M TFA at 100oC for 4 h, followed by reduction with 1% NaBH4 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 separations were performed on two different columns with different temperature as follows;

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。 ¿ - 通过 C18 sep-pak 滤芯消除、脱盐、去除硼酸盐、清洁 O-连接的碳水化合物部分通过 ¿ 从糖肽上裂解下来简而言之，将 1M 硼氢化钠 (NaBH4) 的 50 mM 氢氧化钠 (NaOH) 溶液添加到样品中，并在 45℃ 下孵育 17 小时，然后用 10% 乙酸中和并通过填充柱脱盐。 DOWEXTM 树脂（50W x 8 100，Sigma Aldrich），然后冻干样品。在氮气流下用甲醇:乙酸（9:1，v/v）清除硼酸盐，然后将样品通过 C18 反相柱，首先用 5 洗脱。 % 乙酸，然后用 20% 异丙醇的 5% 乙酸、40% 溶液串联洗脱肽将含 5% 乙酸的异丙醇和 100% 异丙醇分别放入微量离心管中，通过冻干将碳水化合物级分干燥，而将肽级分在快速真空浓缩器中干燥，然后合并在一根管中。 全氧甲基化碳水化合物的制备，通过 C18 sep-pak 柱进行净化 将碳水化合物部分溶解在二甲亚砜中，然后根据Anumula和Taylor的方法(Anumula和Taylor，1992)全甲基化。通过添加水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，将甲基化聚糖装入 C18 sep-pak 柱中，然后用纳米纯水和 15% 洗涤。然后用85％乙腈洗脱纯化的聚糖，并在氮气流下干燥。 糖基连接分析 为了测定糖键，由全甲基化 O-聚糖制备部分甲基化糖醇乙酸酯。 简而言之，将全甲基化聚糖用 2M TFA 在 100℃ 下水解 4 小时，然后用 30mM NaOH 中的 1% NaBH4 还原，并用乙酸酐/吡啶乙酰化。 (1:1，v/v) 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) 在与 5970 MSD（质量选择检测器，电子轰击电离模式）连接的 Hewlett Packard 5890 GC 上分析部分甲基化的糖醇乙酸酯。在不同温度的两个不同柱上进行如下分离；