N-LINKED OLIGOSACCHARIDE PROFILING

N-连接寡糖分析

• 批准号: 8363096
• 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/8363096
• 关键词: 1-Propanol; Acetic Acids; Acetone; Acids; Agitation; Buffers; Centrifugation; Chloroform; Digestion; Freeze Drying; Funding; Glycopeptides; Grant; Heating; Ice; Incubated; Individual; Ions; Kidney; Link; Lipids; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Methanol; Methods; National Center for Research Resources; Nitrogen; Oligosaccharides; Peptide N-glycohydrolase F; Peptides; Polysaccharides; Powder dose form; Preparation; Principal Investigator; Procedures; Propanols; Proteins; Reporting; Research; Research Infrastructure; Resources; Salts; Sampling; Sep-Pak C18; Series; Solutions; Solvents; Source; Speed; Stream; Technology; Temperature; Time; 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: Protein rich powder was prepared from each type of kidneys as described in previous report. N-glycans were released enzymatically by PNGase F and the released N-glycans were permethylated and profiled by mass spectrometry. The detailed procedures are shown below. Preparation of protein rich powder from kidneys Kidneys were homogenized and de-lipidated followed by the method of Aoki.et.al (2007). Briefly, kidneys were homogenized by homogenizer on ice. Lipids were extracted by adjusting the solvent mixture to give a final ratio of chloroform/methanol/water equal to 4:8:3. The extract was incubated at room temperature with end-over-end agitation. The insoluble proteinaceous material was collected by centrifugation and re-extracted three times. The final pellet of insoluble protein was further washed with cold-acetone/water (4:1, v/v) four times and dried under a stream of nitrogen. N-linked glycan preparation The dried 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 and spun at 3000 rpm in a refrigerated centrifuge for 15 minutes. The supernatant was collected and dried. 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 released N-glycans was eluted with 5% acetic acid and dried by lyophilization, and then permethylated based on the method of Anumula and Taylor (Anumula and Taylor, 1992) 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). For the purpose of comparison of peak intensity of each N-glycan component, at least three spectra were obtained for each sample and then intensity % of each N-glycan components among total N-glycans observed in individual spectra were calculated then averaged from the three spectra for each sample.

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： 如先前报告所述，从每种类型的肾脏制备富含蛋白质的粉末。 N-聚糖通过PNGase F酶促释放，释放的N-聚糖被全甲基化并通过质谱分析。 详细程序如下所示。 从肾脏中制备富含蛋白质的粉末 将肾脏均质化并脱脂，然后采用www.example.com（2007）的方法。 简而言之，在冰上通过匀浆器将肾脏匀浆。 通过调节溶剂混合物以得到等于4：8：3的氯仿/甲醇/水的最终比率来提取脂质。 在室温下培养浸提液，并进行上下颠倒搅拌。 通过离心收集不溶性蛋白质材料并再提取三次。 将最终的不溶性蛋白质沉淀进一步用冷丙酮/水（4：1，v/v）洗涤四次，并在氮气流下干燥。 N-连接聚糖制备 将干燥的样品溶解于含有0.01M CaCl2的0.1M 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 ℃下加热5分钟至胰蛋白酶，并在冷冻离心机中以3000 rpm旋转15分钟。 收集上清液并干燥。 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，将样品通过C18 sep-pak柱，用5%乙酸洗脱释放的N-聚糖，并通过冻干干燥，然后根据Anumula和Taylor的方法（Anumula和Taylor，1992）进行全甲基化，并通过质谱法进行分析。 质谱 采用反射器正离子模式，以20mg/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。 为了比较各N-聚糖组分的峰强度，获得各样品的至少三个光谱，然后计算各N-聚糖组分在单个光谱中观察到的总N-聚糖中的强度%，然后根据各样品的三个光谱求平均值。