O-LINKED OLIGOSACCHARIDE PROFILING

O-连接寡糖分析

• 批准号: 8363104
• 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/8363104
• 关键词: Acetic Acids; Acetone; Acids; Agitation; Blood capillaries; Borates; Carbohydrates; Centrifugation; Chloroform; Cleaved cell; Excision; Freeze Drying; Funding; Gases; Grant; Ice; Incubated; Ions; Kidney; Link; Lipids; MALDI-TOF Mass Spectrometry; Maps; Mass Spectrum Analysis; Methanol; Methods; National Center for Research Resources; Nitrogen; Oligosaccharides; Phase; Plant Resins; Polysaccharides; Powder dose form; Preparation; Principal Investigator; Procedures; Proteins; Reporting; Research; Research Infrastructure; Resolution; Resources; Sampling; Scanning; Sep-Pak C18; Solutions; Solvents; Source; Stream; Technology; Temperature; Time; United States National Institutes of Health; Water; base; capillary; chemical reaction; cost; instrument; ion source; mass spectrometer

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 reports. O-glycans were released from protein rich powder by a chemical reaction, which is called ¿-elimination, followed by desalting, borate removal and cleaning up by C18 sep-pak. The released O-glycans were then permethylated and profiled by mass spectrometry. The detailed procedures are shown below. Preparation of protein rich powder from kidneys Kidneys (3 set of kidneys per a sample) 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. O-linked glycan preparation O-linked carbohydrate fractions were cleaved from protein rich powder by ¿-elimination procedures. Briefly, 1 M sodiumborohydride in 50 mM Sodiumhydroxide (NaOH) were added to the samples and incubated overnight at 45oC. The incubated samples were neutralized with 10% acetic acid and desalted by passing through a packed column of DowexTM resins (50 W x 8--100, Sigma Aldrich, St. Louis, MO) and lyophilized. The borate was removed with methanol/acetic acid (9:1) under a stream of nitrogen gas, and the samples were passed through a C18 reversed phase cartridge. The carbohydrate fractions (O-linked glycans) were eluted with 5% acetic acid. The carbohydrate fractions were 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). NSI-MSn analysis was determined by using on a LTQ Orbitrap XL mass spectrometer (ThermoFisher) equipped with a nanospray ion source. Permethylated glycans from each sample were dissolved in the same amount of 1mM NaOH in 50% methanol and infused directly into the instrument at a constant flow rate of 0.5 ¿L/ min. A full FTMS spectrum was collected at 30 000 resolution with 3 microscans. The peak intensities of each O-glycan components were obtained by averaging the 30 full FTMS scans for each sample. The capillary temperature was set at 210oC and MS analysis was performed in the positive ion mode. For total ion mapping (automated MS/MS analysis), m/z range, 300 to 2000 was scanned with ITMS mode in successive 2.8 mass unit windows that overlapped the preceding window by 2 mass units

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。 并且子项目的首席研究员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： 如之前的报告所述，从每种类型的肾脏中制备富含蛋白质的粉末，通过化学反应从富含蛋白质的粉末中释放O-聚糖，该化学反应称为¿ -消除，然后通过 C18 sep-pak 脱盐、去除硼酸盐和净化，然后对释放的 O-聚糖进行全甲基化并通过质谱分析进行分析。详细过程如下所示。 肾脏富含蛋白粉的制备 肾脏（每个样品 3 组肾脏）按照 Aoki.et.al（2007）的方法进行均质化和脱脂。简言之，通过调节溶剂混合物在冰上将肾脏均质化以提取脂质。氯仿/甲醇/水的最终比例等于4:8:3，在室温下彻底搅拌，收集不溶性蛋白质材料。离心并重新提取三次，最后用冷丙酮/水（4:1，v/v）洗涤四次，并在氮气流下干燥。 O-连接聚糖的制备 O-连接碳水化合物部分通过 ¿ 从富含蛋白质的粉末中裂解出来简而言之，将 1 M 硼氢化钠的 50 mM 氢氧化钠 (NaOH) 溶液添加到样品中，并在 45°C 下孵育过夜。用 10% 乙酸中和孵育的样品，并通过 DowexTM 树脂填充柱进行脱盐。 50 W x 8--100，Sigma Aldrich，圣路易斯，密苏里州）并冻干硼酸盐。甲醇/乙酸(9:1)在氮气流下进行，并且样品通过C18反相柱，用5%乙酸洗脱碳水化合物级分。通过冻干，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。 NSI-MSn 分析通过使用配备纳喷雾离子源的 LTQ Orbitrap XL 质谱仪 (ThermoFisher) 进行测定，将来自每个样品的全甲基化聚糖溶解在等量的 1mM NaOH 的 50% 甲醇中，并在 30℃ 下直接注入仪器中。 0.5¿的恒定流量L/分钟以 30 000 分辨率采集了 3 次微扫描，通过平均每个样品的 30 次完整 FTMS 扫描获得了每个 O-聚糖组分的峰强度。毛细管温度设置为 210oC 并进行 MS 分析。在正离子模式下进行。 对于总离子图谱（自动 MS/MS 分析），使用 ITMS 模式在连续 2.8 个质量单位窗口中扫描 m/z 范围 300 至 2000，该窗口与前面的窗口重叠 2 个质量单位