N-LINKED OLIGOSACCHARIDE PROFILING (MASS SPECTROMETRY) OF TWO SAMPLES

两个样品的 N 联寡糖分析（质谱）

• 批准号: 7722654
• 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/7722654
• 关键词: 1-Propanol; 2-Mercaptoethanol; Acetic Acid; Acetic Acids; Acetonitriles; Acids; Buffers; Carbohydrates; Computer Retrieval of Information on Scientific Projects Database; Digestion; Dimethyl Sulfoxide; Freeze Drying; Funding; Gases; Glycol; Glycols; Glycopeptides; Grant; Heating; Ice; Incubated; Institution; Ions; Lasers; Link; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Methanol; Methylene Chloride; Nitrogen; Oligosaccharides; Peptide N-glycohydrolase F; Peptides; Phase; Phosphate Buffer; Polysaccharides; Potassium; Preparation; Propanols; Proteins; Proteomics; Reaction; Research; Research Personnel; Resources; Salts; Sampling; Sep-Pak C18; Series; Solutions; Source; Speed; Stream; Time; Tube; United States National Institutes of Health; Vacuum; Water; methyl iodide; sodium phosphate

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. Release of N-linked glycans The samples (5mg as glycol protein amount of each) were dried and resuspended in 65¿l of nanopure H2O and then 20 ul of 100 mM Sodium phosphate buffer (pH 7.5) and 5ul of denaturing buffer (1% SDS and 1M ¿-mercaptoethanol in Nanopure H2O) were added. The samples then were denatured by heating for 5 minutes at 100¿ C. After cooling, 10 ul of 1M KCl in nanopure H2O was added to each sample solution and then was mixed by vortexing the tube. The sample tubes were placed on ice for 15 minutes and then were spun at maximum speed in a refrigerated microcentrifuge for 10 minutes to pellet the potassium salts of SDS. Ninety microliters of supernatant of each sample were transferred into fresh tube and then 5ul of PNGase F (New English BioLabs) were added to each sample solution, mixed and then incubated at 37oC overnight. After enzymatic digestion, the sample was passed through a C18 reversed phase cartridge. The carbohydrate fraction (N-linked glycan) was eluted first with 5% acetic acid and then the O-linked glycopeptide and peptides was eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and then 100% iso-propanol. The carbohydrate fraction was dried by lyophilization, whereas the peptide fractions were dried in a Speed Vacuum Concentrator and then were combined into one tube. Preparation of the per-O-methylated carbohydrates, cleaning up by C18 The lyophilized carbohydrate fraction was dissolved in dimethylsulfoxide and then methylated with NaOH and methyl iodide (Ciucanu and Kerek, 1984). 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 and were dissolved with methanol prior to analysis by mass spectrometry. 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).

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。 N-连接聚糖的释放 将样品（每个 5mg 作为乙二醇蛋白量）干燥并重悬于 65¿ l 纳米纯 H2O，然后 20 ul 100 mM 磷酸钠缓冲液 (pH 7.5) 和 5ul 变性缓冲液（1% SDS 和 1M ¿-巯基乙醇，溶于 添加 Nanopure H2O)，然后在 100° 下加热 5 分钟使样品变性。 C.冷却后，10ul 将 1M KCl 的纳米纯水添加到每个样品溶液中，然后通过涡旋管混合样品。 将管置于冰上 15 分钟，然后在冷冻微量离心机中以最大速度旋转 10 分钟沉淀 SDS 的钾盐，将每个样品的 90 微升上清液转移到其中。 然后将 5ul PNGase F (New English BioLabs) 添加到每个样品溶液中，混合，然后 37°C 孵育过夜。酶消化后，将样品通过 C18 反相柱。 首先用 5% 乙酸洗脱碳水化合物部分（N-连接聚糖），然后用 O-连接糖肽和 肽依次用 20% 异丙醇的 5% 乙酸溶液、40% 异丙醇的 5% 乙酸溶液洗脱，然后 100% 异丙醇通过冻干干燥碳水化合物级分，而在干燥器中干燥肽级分。 然后将高速真空浓缩器组合成一管。 全氧甲基化碳水化合物的制备，C18 净化 将冻干的碳水化合物部分溶解在二甲亚砜中，然后用 NaOH 和甲基化 碘化物（Ciucanu 和 Kerek，1984）通过添加水和全 O-甲基化来猝灭反应。 用二氯甲烷提取碳水化合物，进一步清除污染物。 简而言之，将聚糖装入 C18 sep pak 柱中，然后用纳米纯水和 15% 然后用85%乙腈洗脱纯化的聚糖，并在气流下干燥。 在进行质谱分析之前将氮气溶解于甲醇中。 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溶液 (50%甲醇:水)作为基质，所有光谱均通过使用 4700 蛋白质组分析仪 (Applied) 获得。 生物系统）。