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。 首先，将样品（~3.5mg）用去离子水在4℃下透析以交换缓冲液。 干燥并溶解在 50mM 柠檬酸钠缓冲液（pH 6.0）中，并进行神经氨酸酶消化。 使用少量等分试样 (~225ug) 进行神经氨酸酶消化、N 连接分析以确认完整 将剩余物干燥并保存在 4 oC 下，然后送回实验室。 下面详细显示。 神经氨酸酶消化 使用纳米纯水在 4 oC 下通过 7.5kDa 截留膜 (Milipore) 透析样品 (~3.5mg) 过夜 去除盐分，然后在 Speed Vac 中干燥。用 50mM 柠檬酸钠缓冲液重新配制样品。 (pH 6.0) 和 Neuroraminidase (New England Biolabs) 添加到样品中并在 37 ¿ C过夜。 N-连接聚糖分析 <通过丙酮沉淀去除污染物> 将丙酮:水(4:1)添加到干燥的样品中，将样品溶液置于冰上15分钟，然后。 在冷冻微量离心机中以最大速度旋转 15 分钟以沉淀上清液。 取出样品两次。 <N-连接聚糖的释放和全甲基化> 将样品重悬于 20mM 磷酸钠缓冲液 (pH 7.5) / 0.05%SDS, 50mM ¿ -巯基乙醇。 然后在 100°C 下加热 5 分钟使样品变性。冷却后，将 10% (v/v) 的 1M KCl 添加到样品中。 将溶液和样品管置于冰上 15 分钟，然后在离心机中以最大速度离心。 冷冻微量离心机 10 分钟沉淀上清液中的钾盐，然后将其转移至离心机中。 然后将新鲜试管中的 PNGase F (New England BioLabs) 添加到样品中，然后在 37 oC 下孵育过夜。 酶消化后，样品通过 C18 反相柱，碳水化合物组分 (N-连接聚糖)用5％乙酸洗脱并通过冷冻干燥将由此获得的N-连接聚糖进行干燥。 根据 Anumula 和 Taylor 的方法（Anumula 和 Taylor，1992）进行全甲基化，并通过 MALDI-TOF/MS 进行分析。 <基质辅助激光解吸飞行时间质谱分析法(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，使用4700蛋白质组分析仪(Applied)获得光谱。 生物系统）。