N-LINKED OLIGOSACCHARIDE PROFILING

N-连接寡糖分析

• 批准号: 8363103
• 负责人: Parastoo Azadi
• 金额: $ 0.17万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363103
• 关键词: 1-Propanol; Acetic Acids; Acetonitriles; Acids; Buffers; Carbohydrates; Digestion; Dimethyl Sulfoxide; Freeze Drying; Funding; Gases; Glycopeptides; Grant; Heating; Incubated; Ions; Link; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Methanol; Methods; Methylene Chloride; National Center for Research Resources; Nitrogen; Oligosaccharides; Peptide N-glycohydrolase F; Peptides; Polysaccharides; Preparation; Principal Investigator; Procedures; Propanols; Reaction; Research; Research Infrastructure; Resources; Salts; Sampling; Sep-Pak C18; Series; Solutions; Source; Speed; Stream; Technology; Trypsin; United States National Institutes of Health; Vacuum; Water; base; cost; sugar

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Methods: First of all, the sample was denatured and then digested with Trypsin followed by glycopeptides enrichment. N-linked glycans were then released enzymatically by PNGase F. Released glycans were separated by C18 sep-pak and permethylated prior to mass spec analysis. Detailed procedures used for your sample analysis are shown in detail below. Release of N-linked glycans The sample was dissolved in 0.1 M Tris-HCl buffer, pH 8.2 containing 0.01 M 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). The sample was then heated at 100¿ C for 5 min to inactivate trypsin. The sample was then passed through a C18 sep-pak cartridge and washed with 5% acetic acid to remove contaminants (salts, free sugar, etc.). Peptides and glycopeptides were eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and 100% iso-propanol and dried in a speed vacuum concentrator. The dried samples were combined and incubated with PNGase F at 37¿ C overnight to release N-glycans. After digestion, the sample was passed through a C18 sep-pak cartridge and the carbohydrate fraction was eluted with 5% acetic acid and dried by lyophilization. Preparation of the per-O-methylated carbohydrates The carbohydrate fraction was dissolved in dimethylsulfoxide and then permethylated based on the method of Anumula and Taylor (Anumula and Taylor, 1992). 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 and profiled by mass spectrometry. Mass spectrometry 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 Microflex LRF (Bruker).

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 研究方法： 首先将样品变性，然后用胰蛋白酶消化，接着进行糖肽富集。 然后通过PNGase F酶促释放N-连接聚糖。 通过C18 sep-pak分离释放的聚糖，并在质谱分析前进行全甲基化。 用于样品分析的详细程序如下所示。 N-连接聚糖的释放 将样品溶解于含有0.01 M CaCl 2的0.1 M Tris-HCl缓冲液（pH 8.2）中。 然后通过在100 ℃下加热5分钟使样品变性。 冷却后，用胰蛋白酶消化样品（37 ℃，过夜）。 然后将样品在100 ° C下加热5分钟至胰蛋白酶。 然后将样品通过C18 sep-pak柱，并用5%乙酸洗涤以除去污染物（盐、游离糖等）。 用20%异丙醇的5%乙酸溶液、40%异丙醇的5%乙酸溶液和100%异丙醇连续洗脱肽和糖肽，并在高速真空浓缩器中干燥。 将干燥的样品合并，并与PNGase F在37 ℃下孵育过夜以释放N-聚糖。 消化后，使样品通过C18 sep-pak柱，用5%乙酸洗脱碳水化合物级分，并通过冻干干燥。 全-O-甲基化碳水化合物的制备 将碳水化合物部分溶于二甲亚砜中，然后根据Anumula和Taylor的方法（Anumula和Taylor，1992）进行全甲基化。 通过加入水淬灭反应，用二氯甲烷萃取全-O-甲基化的碳水化合物。 进一步清除全-0-甲基化聚糖的污染物。 简言之，将聚糖加载到C18 sep pak柱中，然后用纳米纯水和15%乙腈洗涤。 然后用85%乙腈洗脱聚糖。 将纯化的聚糖在氮气流下干燥，用甲醇溶解，并通过质谱分析。 质谱 采用反射器正离子模式，以20 mg/mL的DHBA（50%甲醇：水）为基质，进行MALDI/TOF-MS。通过使用Microflex LRF（Bruker）获得光谱。