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： 首先，通过向溶液中添加氯仿和甲醇来提取脂质并从发酵溶液中沉淀蛋白质。将沉淀物进一步用丙酮/水溶液洗涤四次，使用少量等分的富含蛋白质的粉末(1%)来检查。通过SDS-PAGE对蛋白粉末进行b-消除，以进行O-聚糖分析。然后将b-消除后的溶液通过Dowex树脂脱盐，然后进行脱盐。通过硼酸盐去除和 C18 净化，对由此获得的释放的 O-聚糖进行全甲基化并通过质谱分析进行分析。用于样品分析的详细程序如下所示。 由发酵液制备富含蛋白质的粉末 根据Aoki.等人(2007)的方法从发酵溶液中制备富含蛋白质的粉末。简言之，通过调节溶剂混合物以得到氯仿​​/甲醇/的最终比例，除去脂质并从发酵溶液中沉淀蛋白质。水等于4:8:3，在室温下温育，通过离心收集不溶性蛋白质物质，并重新提取3次。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。 聚丙烯酰胺凝胶电泳 通过 SDS-PAGE 分析等份富含蛋白质的粉末 (1%)，以了解富含蛋白质的粉末中的蛋白质含量。电泳后，将分离的蛋白质用 4-15% SDS-PAGE 梯度凝胶分离。考马斯蓝。 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言之，将 1 mL 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，并通过填充柱脱盐。 DowexTM 树脂（50 W x 8--100，Sigma Aldrich，圣路易斯，密苏里州）并冻干。除去硼酸盐。在氮气流下用甲醇/乙酸(9:1)洗脱，并将样品通过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 AB SCIEX TOF/TOF" 5800 系统（Applied Biosystems）获得光谱。 NSI-MSn 分析按照复杂碳水化合物研究中心开发的方法进行（Aoki 等人，2007 年），使用配备全甲基化聚糖的 LTQ Orbitrap XL 质谱仪 (ThermoFisher) 进行测定。将其溶解在 50% 甲醇中的 1mM NaOH 中，并以 0.5 ¿ 的恒定流速直接注入仪器中L/分钟以 30 000 分辨率收集完整的 FTMS 谱图，毛细管温度设置为 210°C，并在正离子模式下进行 MS 分析。 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 800 至 2000，该窗口与前面的窗口重叠 2 个质量单位。