STRUCTURAL ANAYSIS OF N-LINKED OLIGOSACCHRIDES

N-连接寡糖的结构分析

• 批准号: 7722669
• 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/7722669
• 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; Glycopeptides; Grant; Heating; Ice; Incubated; Institution; Ions; Lasers; Link; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Methanol; Methylation; Methylene Chloride; New England; Nitrogen; Oligosaccharides; Peptide N-glycohydrolase F; Peptides; Phase; Phosphate Buffer; Polysaccharides; Potassium; Propanols; Protein Denaturation; Proteomics; Reaction; Research; Research Personnel; Resources; Salts; Sampling; Sep-Pak C18; Series; Solutions; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; 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 were transferred to microcentrifuge tubes and dried in a vacuum centrifuge. The dried samples were resuspended in 65 ¿L of nanopure H2O and then 20 ¿L of 100 mM sodium phosphate buffer (pH 7.5) and 5 ¿L of denaturing buffer (1% SDS and 1 M ¿-mercaptoethanol in nanopure H2O) were added. Denaturation of protein was accomplished by heating for 5 min at 100oC. After cooling, 10 ¿L 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 min and then were spun at maximum speed in a refrigerated microcentrifuge for 10 min to pellet the potassium salts of SDS. Ninety microliters of supernatant of each sample were transferred into another tube and then 5 ¿L of PNGase F (New England BioLabs) were added to each sample solution (supernatant), 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 fraction was eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and then 100% iso-propanol. Since both samples were less than 5 mg (4611-154A, 1.5 mg; 4611-154B, 3.1 mg), about 60% of the N-linked fraction was allocated for permethylation. The carbohydrate fractions were dried by lyophilization, whereas the peptide fractions were dried in a vacuum centrifuge and then were combined into one microcentrifuge tube. Per-O-methylation of carbohydrates and purification by C18 sep-pak cartridge The lyophilized eluate intended for N-linked oligosaccharide profiling 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 dissolved in 1:1 methanol:water and loaded into a C18 sep pak cartridge and then washed with nanopure water. Per-O-methyl carbohydrates were eluted with 15% acetonitrile into a screw-cap tube, and with 85% acetonitrile into another screw-cap tube. The glycans eluted with 85% acetonitrile were dried under a stream of nitrogen gas and were dissolved with methanol for analysis by mass spectrometry. Matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF) Profiling of N-linked glycans was performed by MALDI/TOF mass spectrometry. The machine used was a 4700 Proteomics analyzer (Applied Biosystems), which was set in the reflector positive ion mode. Permethylated glycans were crystallized on a MALDI plate with ¿-dihydroxybenzoic acid (DHBA, 20 mg/mL solution in 50% methanol:water) as a matrix.

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 将样品转移到微量离心管中并在真空离心机中干燥，将干燥的样品重新悬浮在65°C中。 L 纳米纯 H2O，然后 20 ¿ L 100 mM 磷酸钠缓冲液 (pH 7.5) 和 5 ¿添加 L 变性缓冲液（1% SDS 和 1 M 巯基乙醇在纳米纯水中），通过在 100°C 下加热 5 分钟来完成蛋白质变性，然后冷却 10°C。将 L 1M KCl 的纳米纯水添加到每个样品溶液中，然后通过涡旋管将样品管置于冰上 15 分钟，然后在冷冻微量离心机中以最大速度旋转 10 分钟以沉淀钾。将每个样品的 90 微升上清液转移到另一个管中，然后将 5 ¿将 L 的 PNGase F（New England BioLabs）添加到每个样品溶液（上清液）中，混合，然后在 37oC 下孵育过夜。酶消化后，将样品通过 C18 反相柱，得到碳水化合物组分（N-连接聚糖）。 ）首先用 5% 乙酸洗脱，然后用 20% 异丙醇串联洗脱 O-连接糖肽和肽级分。 5% 乙酸、5% 乙酸中的 40% 异丙醇，然后是 100% 异丙醇，因为两个样品均小于 5 mg（4611-154A，1.5 mg；4611-154B，3.1 mg），约为 60%。 N-连接级分被分配用于全甲基化，碳水化合物级分通过冻干进行干燥，而碳水化合物级分被分配用于全甲基化。将肽级分在真空离心机中干燥，然后合并到一根微量离心管中。 碳水化合物的全氧甲基化和 C18 sep-pak 柱纯化 将用于 N-连接寡糖分析的冻干洗脱液溶解在二甲亚砜中，然后用 NaOH 和甲基碘进行甲基化（Ciucanu 和 Kerek，1984）。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，将全O-甲基化聚糖溶解在水中。将 1:1 甲醇:水装入 C18 sep pak 柱中，然后用纳米纯水洗涤，用 15% 乙腈将全氧甲基碳水化合物洗脱到螺旋盖管中，并用 85% 乙腈洗脱到另一个螺旋盖中。用85%乙腈洗脱的聚糖在氮气流下干燥，并用甲醇溶解用于质谱分析。 基质辅助激光解吸飞行时间质谱 (MALDI-TOF) 通过 MALDI/TOF 质谱法对 N-连接聚糖进行分析，所使用的机器是 4700 蛋白质组分析仪（Applied Biosystems），其设置为反射器正离子模式，并在 MALDI 板上结晶。 -二羟基苯甲酸（DHBA，20 mg/mL 50% 甲醇:水溶液）作为基质。