O-LINKED OLIGOSACCHARIDE PROFILING

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： 如先前报告中所述，从每种类型的肾脏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出O-聚糖，该化学反应被称为消除，随后是脱羧、硼酸盐去除和C18 sep-pak的清洁。 然后将释放的O-聚糖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（每个样品3组肾脏）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。 O-连接聚糖制备 通过消除程序从富含蛋白质的粉末中裂解O-连接的碳水化合物级分。 简言之，将溶于50 mM氢氧化钠（NaOH）的1 M硼氢化钠加入样品中，并在45 ℃下孵育过夜。用10%乙酸中和温育的样品，通过DowexTM树脂填充柱（50 W × 8 - 100，Sigma Aldrich，St.Louis，MO）脱盐，并冻干。 在氮气流下用甲醇/乙酸（9：1）除去硼酸盐，并使样品通过C18反相柱。 用5%乙酸洗脱碳水化合物级分（O-连接聚糖）。 通过冻干干燥碳水化合物级分，然后基于Anumula和Taylor的方法（Anumula和Taylor，1992）进行全甲基化，并通过质谱分析。 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。 通过使用配备有纳米喷雾离子源的LTQ Orbitrap XL质谱仪（ThermoFisher）测定NSI-MSn分析。 将每个样品的全甲基化聚糖溶解在相同量的1mM NaOH的50%甲醇溶液中，并以0.5 μ L/min的恒定流速直接注入仪器中。使用3次显微扫描以30000分辨率收集完整的FTMS光谱。 通过对每份样品的30次完整FTMS扫描求平均值，获得各O-聚糖组分的峰强度。 毛细管温度设定为210 ℃，以正离子模式进行MS分析。 对于总离子图（自动MS/MS分析），在与前一窗口重叠2个质量单位的连续2.8质量单位窗口中，使用ITMS模式扫描m/z范围300至2000

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