DETERMINATION OF N-TERMINUS SEQUENCE AND O-LINKED GLYCAN ANALYSIS

N 末端序列的测定和 O 连接聚糖分析

• 批准号: 8363082
• 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/8363082
• 关键词: Acetic Acids; Acetone; Acids; Agitation; Aliquot; Blood capillaries; Borates; Carbohydrates; Centrifugation; Chloroform; Cleaved cell; Complex; Coomassie blue; Electrophoresis; Excision; Fermentation; Freeze Drying; Funding; Gases; Gel; Grant; Incubated; Ions; Link; Lipids; MALDI-TOF Mass Spectrometry; Maps; Mass Spectrum Analysis; Methanol; Methods; National Center for Research Resources; Nitrogen; Phase; Plant Resins; Polysaccharides; Powder dose form; Preparation; Principal Investigator; Procedures; Proteins; Research; Research Infrastructure; Resolution; Resources; Rest; Sampling; Scanning; Solutions; Solvents; Source; Staining method; Stains; Stream; System; Technology; Temperature; Time; United States National Institutes of Health; Water; base; capillary; cost; instrument; ion source; mass spectrometer; water solution

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: First, lipids were extracted and proteins were precipitated from fermentation solution by adding chloroform and methanol to the solution. The precipitate was further washed with acetone/water solution four times. A small aliquot of protein rich powder (1%) was used for examine the protein powder by SDS-PAGE and the rest of the powder was subjected to b-elimination for O-glycan analysis. The solution after b-elimination was then desalted by passing though Dowex resin, followed by borate removal and C18 clean up. The released O-glycans thus obtained were permethylated and profiled by mass spectrometry. The detailed procedures used for your sample analysis are shown in detail below. Preparation of protein rich powder from fermentation solution Protein rich powder was prepared from fermentation solution according to the method of Aoki.et.al (2007). Briefly, lipids were removed and proteins were precipitated from the fermentation solution 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. SDS-PAGE An aliquot of protein rich powder (1%) were analyzed by SDS-PAGE to see protein content in protein rich powder. The samples were separated in a 4-15% SDS-PAGE gradient gel. After electrophoresis, the resolved proteins were stained with Coomassie blue. O-linked glycan preparation O-linked carbohydrate fractions were cleaved from protein rich powder by ¿-elimination procedures. Briefly, 1 mL of 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 streamof 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 AB SCIEX TOF/TOF" 5800 System (Applied Biosystems). NSI-MSn analysis was performed following the method developed at the Complex Carbohydrates Research Center (Aoki et. al, 2007). Mass analysis was determined by using on a LTQ Orbitrap XL mass spectrometer (ThermoFisher) equipped with a nanospray ion source. Permethylated glycans were dissolved in 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. 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, 800 to 2000 was scanned with ITMS mode in successive 2.8 mass unit windows that overlapped the preceding window by 2 mass units.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 方法： 首先，通过在溶液中添加氯仿和甲醇，从发酵溶液中提取脂质并从发酵溶液中精确。将沉淀物进一步用丙酮/水溶液清洗四次。使用SDS-PAGE的少量等分试样富含蛋白质的粉末（1％）来检查蛋白质粉末，其余的粉末进行了B-溶解以进行O-聚糖分析。然后通过通过Dowex树脂进行脱盐后，将溶液脱盐，然后清除硼酸盐，并清理C18。因此获得的释放的O-聚糖被泛甲基化，并通过质谱法进行了分析。用于您的样本分析的详细过程如下所示。 从发酵溶液中制备富含蛋白质的粉末 根据Aoki.et.al（2007）的方法，从发酵溶液中制备富含蛋白质的粉末。简而言之，除去脂质，并通过调节溶液混合物以获得等于4：8：3的氯仿/甲烷/水的最终比例，从而从发酵溶液中保存蛋白质。将提取物在室温下以终端搅拌孵育。通过离心收集不溶性的蛋白质材料并重新提取了三次。不溶性蛋白的最终颗粒被冷丙酮/水（4：1，v/v）进一步洗涤，并在氮流下干燥。 SDS-PAGE 通过SDS-PAGE分析了富含蛋白质粉末的等分试样（1％），以查看富含蛋白质粉末中的蛋白质含量。样品在4-15％的SDS-PAGE梯度凝胶中分离。电泳后，将溶解的蛋白质用coomassie蓝色染色。 O连接的聚糖制备 O连接的碳水化合物分数通过``省略者''从富含蛋白质的粉末中裂解。简而言之，将1 mL的1 m钠氢氢氢化物（NaOH）添加到样品中，并在45oC中孵育过夜。将孵育的样品用10％乙酸中和，并通过穿过Dowextm树脂（50 W x 8---100，Sigma Aldrich，St.Louis，Mo）的堆积柱并进行脱盐并脱水。用氮气中的甲醇/乙酸（9：1）除去硼酸盐，并通过C18反向相墨盒通过样品。用5％乙酸洗脱了碳含水量的分数（O连接的聚糖）。通过冻干将碳水化合物分数干燥，然后根据Anumula和Taylor的方法（Anumula and Taylor，1992）干燥，并通过质谱法进行了分析。 质谱法 MALDI/TOF -MS在反射器正离子模式下使用�-二羟基苯甲酸（DHBA，20mg/ml溶液中的50％甲烷：水）作为基质进行。通过使用AB Sciex TOF/TOF“ 5800系统（Applied Biosystems）获得频谱。 根据在复杂的碳水化合物研究中心开发的方法进行NSI-MSN分析（Aoki等，2007）。通过在配备有纳米喷雾离子源的LTQ Orbitrap XL质谱仪（Thermofisher）上使用质量分析。将苄氨基甲基化的糖溶解在1mm NaOH中50％甲醇中，并以0.5 l/min的恒定流速直接注入仪器中。以30 000分辨率收集了完整的FTMS频谱。在210oC处设置毛细管温度，并在正离子模式下进行MS分析。 对于总离子映射（自动MS/MS分析），M/z范围为800至2000，在成功的2.8个质量单位窗口中扫描了ITMS模式，该窗口与前面窗口重叠的2.8个质量单位窗口与2个质量单位。