EXOGLYCOSIDASE DIGESTION OF ONE SAMPLE

一份样品的外糖苷酶消化

• 批准号: 7956035
• 负责人: Parastoo Azadi
• 金额: $ 0.13万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7956035
• 关键词: 2-Mercaptoethanol; Acetic Acids; Acetone; Acids; Aliquot; Back; Buffers; Carbohydrates; Centrifugation; Computer Retrieval of Information on Scientific Projects Database; Dialysis procedure; Digestion; Excision; Exoglycosidases; Freeze Drying; Funding; Grant; Heating; Ice; Incubated; Institution; Ions; Lasers; Link; Mass Spectrum Analysis; Membrane; Methanol; Methods; Neuraminidase; New England; Peptide N-glycohydrolase F; Phase; Phosphate Buffer; Polysaccharides; Potassium; Precipitation; Procedures; Proteins; Proteomics; Research; Research Personnel; Resources; Salts; Sampling; Solutions; Source; Speed; Technology; Time; Tube; United States National Institutes of Health; Water; base; reconstitution; sodium citrate; 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. First of all, the sample (~3.5mg) was dialyzed with deionized water at 4 oC to exchange buffer. The sample were then dried and dissolved in 50mM Sodium Citrate buffer (pH 6.0) and Neuroraminidase digestion was performed. After Neuraminidase digestion, N-linked profiling was performed using a little aliquot (~225ug) to confirm complete exoglycosidase digestion. The remainder was dried and stored at 4 oC and then sent back to your lab. The procedures are shown in detail below. Neuroraminidase digestion The sample (~3.5mg) was dialyzed with a 7.5kDa cut-off membrane (Milipore) using nanopure water at 4 oC overnight to remove salts and then dried down in a Speed Vac. The sample was reconstituted with 50mM Sodium Citrate buffer (pH 6.0) and Neuroraminidase (New England Biolabs) is added to the sample and incubated at 37 ¿C overnight. N-linked Glycan analysis <Removal of contaminants by Acetone precipitation> Acetone:Water (4:1) was added to the dried sample. The sample solution was placed on ice for 15 minutes and then spun at maximum speed in a refrigerated microcentrifuge for 15 minutes to pellet the protein. The supernatant was removed. The sample was washed twice. <Release and permethylation of N-linked glycans> The sample was resuspended in 20mM Sodium phosphate buffer (pH 7.5) / 0.05%SDS, 50mM ¿-mercaptoethanol. The sample was then denatured by heating for 5 minutes at 100o C. After cooling, 10% (v/v) of 1M KCl was added to the solution and the sample tube was placed on ice for 15 minutes followed by centrifugation at maximum speed in a refrigerated microcentrifuge for 10 minutes to pellet the potassium salts of SDS. Supernatant was transferred into a fresh tube and then PNGase F (New England BioLabs) was added to the sample and then incubated at 37 oC overnight. After enzymatic digestion, the sample was passed through a C18 reversed phase cartridge and the carbohydrate fraction (N-linked glycan) was eluted with 5% acetic acid and dried by lyophilization. The N-linked glycans thus obtained were permethylated based on the method of Anumula and Taylor (Anumula and Taylor, 1992) and profiled by MALDI-TOF/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. 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。 首先，将样品（〜3.5mg）用4 oC的去离子水透析以交换缓冲液。然后样本是 干燥并溶解在50mm柠檬酸钠缓冲液（pH 6.0）中，并进行了神经胺酶消化。后 使用少量等分试样（〜225UT）进行神经氨酸酶消化，N连锁分析以确认完成 外糖苷酶消化。将其余部分干燥并存储在4 OC中，然后寄回您的实验室。程序 如下所示。 神经胺酶消化 使用纳米水在4 oC中透析样品（〜3.5mg）用7.5kDa截止膜（米利波尔）过夜 取消销售，然后在速度Vac中干燥。样品用50mm柠檬酸钠缓冲液重构 （pH 6.0）和神经氨基酶（新英格兰生物群）被添加到样品中，并在37°C下孵育过夜。 N连接的聚糖分析 <通过丙酮沉淀去除污染物> 丙酮：将水（4：1）添加到干燥的样品中。将样品溶液放在冰上15分钟，然后将 以最大速度旋转在冷藏的微量离心机中15分钟，以颗粒蛋白质。上清液是 删除。样品洗涤两次。 <N连接聚糖的释放和苄苄氨基化> 将样品重悬于20mm磷酸钠缓冲液（pH 7.5） / 0.05％SDS，50mm€-Mercaptoethanol中。这 然后通过在100o C上加热5分钟来变性。冷却后，将10％（v/v）添加到1M KCl中 溶液和样品管在冰上放置15分钟，然后以最大速度离心 冷藏微量离心机10分钟，使SDS的钾盐颗粒。上清液被转移到 将新鲜管，然后将PNGase F（新英格兰Biolabs）添加到样品中，然后在37 oC中孵育过夜。 酶促消化后，样品通过C18反向相墨盒和碳含量分数。 （N连接的聚糖）用5％乙酸洗脱，并通过冻干干燥。因此获得的N连接糖含量为 基于Anumula和Taylor的方法（Anumula and Taylor，1992），并由Maldi-Tof/MS进行了苄苄化化。 <矩阵辅助激光解吸飞机质谱（MALDI-TOF）> 使用 - 二羟基苯甲酸（DHBA，20mg/ml溶液）在反射器正离子模式下进行MALDI/TOF -MS 在50％甲烷：水中）作为基质。通过使用4700蛋白质组学分析仪获得频谱（应用 生物系统）。