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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供 该子项目的主要支持。 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源的子项目可能列出的总成本。 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 方法： 如先前报告所述，从每种类型的肾脏中制备富含蛋白质的粉末，并通过 PNGase F 酶促释放 N-聚糖，并通过质谱分析对释放的 N-聚糖进行全甲基化和分析。 肾脏富含蛋白粉的制备 简言之，按照 Aoki.et.al (2007) 的方法对肾脏进行均质化和脱脂，通过调节溶剂混合物以得到氯仿​​/甲醇/水的最终比例来提取脂质。等于4:8:3，在室温下温育并彻底搅拌，通过离心收集不溶性蛋白质材料并重新提取。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 将干燥的样品溶解在含有 0.01 M CaCl2 的 0.1 M Tris-HCl 缓冲液（pH 8.2）中，然后在 100° 下加热 5 分钟使样品变性。 C. 冷却后，用胰蛋白酶消化样品（37℃，过夜），然后将样品加热至 100°C。 C 5 分钟以灭活胰蛋白酶，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% 乙酸洗涤以去除污染物。肽和糖肽用 20% 异丙醇串联洗脱。 5% 乙酸、40% 异丙醇溶于 5% 乙酸和 100% 异丙醇中，并在快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。 C过夜释放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-聚糖并冻干，然后根据Anumula和的方法进行全甲基化。 Taylor（Anumula 和 Taylor，1992）并通过质谱分析进行分析。 质谱分析 MALDI/TOF-MS 在反射器正离子模式下使用 ¿ -二羟基苯甲酸（DHBA，20mg/mL 50%甲醇：水溶液）作为基质，使用 Microflex LRF (Bruker) 获得光谱。 为了比较每种N-聚糖组分的峰强度，每个样品至少获得三个光谱，然后计算各个光谱中观察到的总N-聚糖中每种N-聚糖组分的强度％，然后对三个光谱进行平均每个样品的光谱。