N-LINKED OLIGOSACCHARIDE PROFILING

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 方法： 按照先前报告的描述，从每种类型的肾脏中制备富含蛋白质的粉末。用PNGase F酶催化N-糖链的释放，并对释放的N-糖链进行全甲基化和质谱分析。具体步骤如下所示。 从肾脏中制备富含蛋白质的粉末 肾脏匀浆和脱脂后采用Aoki.et.al(2007)的方法。简单地说，肾脏在冰上用匀浆器匀浆。通过调整溶剂混合物来提取脂质，最终得到氯仿/甲醇：水的比例为4：8：3。将提取物在室温下进行端-端搅拌。用离心法收集不溶的蛋白质物质，再提取3次。最后的不溶性蛋白质颗粒用冷丙酮/水(4：1，v/v)进一步洗涤4次，并在氮气气流下干燥。 N-连接葡聚糖制剂 干燥样品在0.1M Tris-HCl缓冲液中溶解，pH 8.2，含0.01M CaCl2。然后将样品在100℃加热5分钟变性，冷却后，用胰酶(37℃，过夜)消化样品。然后将样品在100℃下加热5分钟，使胰酶失活，并在冷冻离心机中以3000 rpm旋转15分钟。收集上清液并烘干。然后将样品通过C18 Sep-pak色谱柱，并用5%的冰醋酸洗涤以去除污染物(盐、游离糖等)。多肽和糖肽用20%异丙醇5%冰醋酸、40%异丙醇5%冰醋酸和100%异丙醇串联洗脱，在高速真空浓缩器中干燥。将干燥的样品与PNGase F在37℃下孵育一夜，释放N-糖链。消化后，样品通过C18Sep-pak小柱，释放的N-糖链用5%的冰醋酸洗脱，冷冻干燥，然后根据Anumula和Taylor(Anuula and Taylor，1992)的方法进行全甲基化，并进行质谱分析。 质谱学 MALDI/TOF-MS色谱柱为反射镜正离子模式，以二羟基苯甲酸(DHBA，20 mg/mL甲醇：水)为基质。光谱是用Microflex LRF(Bruker)获得的。 为了比较每个N-糖组分的峰强度，每个样品至少获得三个谱图，然后计算每个谱图中每个N-糖组分在总N-糖链中的强度百分比，然后根据每个样品的三个谱图求平均值。

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.