N-LINKED OLIGOSACCHARIDE PROFILING OF ONE SAMPLE

一个样品的 N 联寡糖分析

• 批准号: 7722676
• 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/7722676
• 关键词: 1-Propanol; Acetic Acid; Acetic Acids; Acetonitriles; Acids; Air; Buffers; Carbohydrates; Computer Retrieval of Information on Scientific Projects Database; Digestion; Dimethyl Sulfoxide; Freeze Drying; Funding; Gases; Glycopeptides; Grant; Heating; Institution; Ions; Lasers; Link; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Methanol; Methylene Chloride; New England; Nitrogen; Oligosaccharides; Peptide N-glycohydrolase F; Peptides; Phase; Phosphate Buffer; Polysaccharides; Preparation; Propanols; Proteomics; Reaction; Research; Research Personnel; Resources; Salts; Sampling; Sep-Pak C18; Series; Solutions; Source; Stream; Temperature; Time; Trypsin; Tube; United States National Institutes of Health; Water; cold temperature; methyl iodide; reconstitution; 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 dried sample was dissolved in Trypsin buffer (0.1M Tris-HCl, pH 8.2 containing 0.01M CaCl2). The sample then was denatured by heating for 5 minutes at 100¿C. After cooling, the sample was digested with the trypsin (37oC, overnight). After tryptic digestion, the sample was heated at 100¿ C for 5 minutes to de-activate the trypsin. After cooling to room temperature, the sample was applied to a C18 sep-pak cartridge. Before elution of glycopeptides and peptides, the sample adsorbed in the C18 sep-pak cartridge was cleaned with 5% acetic acid to remove any possible contaminants (salts, etc.). Peptides and glycopeptides then 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 propanol fractions were dried and then combined in one tube, and then reconstituted with 50mM sodium phosphate buffer (pH 7.5), and then the N-glycans were released using PNGase F (New England BioLabs). After digestion, the sample was passed through a C18 reversed phase cartridge. The carbohydrate fraction (N-linked glycan) was first eluted with 5% acetic acid and then the O-linked glycopeptides and peptides were eluted with 2-PrOH. The carbohydrate fraction was dried by lyophilization, whereas the other fraction was dried under a stream of air at low temperature. Preparation of the per-O-methylated carbohydrates 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. The spectrum was obtained by using a 4700 Proteomics analyzer (Applied Biosystems).

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 释放N连接的聚糖 将干燥的样品溶解在胰蛋白酶缓冲液中（0.1M Tris-HCl，pH 8.2含0.01m CaCl2）。然后通过在100°C的100分钟加热5分钟来变性。冷却后，用胰蛋白酶（37oC，隔夜）消化样品。胰蛋白酶消化后，将样品在100°C加热5分钟以使胰蛋白酶脱离活化。冷却至室温后，将样品应用于C18 Sep-pak墨盒。在洗脱糖肽和在C18 Sep-pak弹药筒中的糖肽之前，用5％乙酸清洁，以去除任何可能的污染物（盐等）。然后将肽和糖肽在5％乙酸中用20％等丙醇串联洗脱，在5％乙酸中40％ISO-丙醇和100％Iso-丙醇分别为微离心管。将丙醇馏分干燥，然后在一个管中混合，然后用50mm磷酸钠缓冲液（pH 7.5）重构，然后使用PNGase F（新英格兰Biolabs）释放N-聚糖。消化后，样品通过C18反向相墨盒。首先用5％的乙酸洗脱碳水化合物馏分（N-连接的聚糖），然后用O-连接的糖肽和肽用2-ProH洗脱。通过冻干使碳水化合物的馏分干燥，而另一部分在低温下的空气流下干燥。 每甲基化碳液的制备 将冻干的碳水化合物馏分溶解在二甲基硫氧化氢中，然后用NaOH和碘化甲基甲基化（Ciucanu and Kerek，1984）。通过添加水和每甲基化的碳素用二氯甲烷提取反应。将 - 甲基化的甘氨酸进一步清洁污染物。简而言之，将聚糖加载到C18 Sep Pak弹药筒中，然后用纳米水和15％的乙腈洗涤。然后用85％的乙腈洗脱聚糖。将纯化的聚糖在氮气流下干燥，并在通过质谱分析之前用甲醇溶解。 基质辅助激光解吸时间飞行时间质谱（MALDI-TOF） MALDI/TOF -MS以反射剂阳性离子模式（使用二羟基苯甲酸（DHBA，20mg/ml溶液）在50％甲醇：水）中作为基质进行。通过使用4700蛋白质组学分析仪（Applied Biosystems）获得光谱。