MALDI/TOF-MS ANALYSIS OF 6 SAMPLES

6 个样品的 MALDI/TOF-MS 分析

• 批准号: 8363076
• 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/8363076
• 关键词: Acetates; Acetic Acids; Acetylation; Acids; Aliquot; Blood capillaries; Carbohydrates; Citrates; Deposition; Dimethyl Sulfoxide; Electrons; Ethanol; Funding; Gases; Grant; Hydrogen; Ions; Maps; Mass Spectrum Analysis; Methanol; Methods; Methylene Chloride; National Center for Research Resources; Phase; Polysaccharides; Preparation; Principal Investigator; Procedures; Reaction; Research; Research Infrastructure; Resolution; Resources; Rest; Sampling; Scanning; Silicon Dioxide; Solutions; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sugar Alcohols; Technology; Temperature; United States National Institutes of Health; Water; Width; acetic anhydride; base; capillary; cost; detector; genetic linkage analysis; instrument; ion source; ionization; mass spectrometer; programs; pyridine; sugar

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: For MALDI analysis, an aliquot (~2ul) of each sample was analyzed following the same method used for previous samples (PR010510Z-A). For glycosyl linkage analysis, 100 ul of sample B, those have been treated with and without laforin, were permethylated. An aliquot of the permethylated sample (~5%) was analyzed by MALDI and ESI. The rest of the permethylated materials were hydrolyzed, reduced and acetylated. The partially methylated alditol acetates thus obtained were profiled by GC-MS. Detailed procedures used for your sample analysis are described below. Glycosyl linkage analysis 1) Preparation of the per-O-methylated carbohydrates The sample was permethylated for the glycosyl linkage analysis. Briefly, the sample was dissolved in dimethylsulfoxide and then permethylated based on the method of Anumula and Taylor (Anumula and Taylor, 1992) and the reaction was quenched by addition of water and per-O-methylated carbohydrates were extracted with dichloromethane. An aliquot of permethylated glycans were examined by MALDI and ESI for confirming complete permethylation. 2) Preparation of partially methylated alditol acectates For determination of sugar linkages, partially methylated alditol acectates were prepared from fully permethylated glycans. Briefly, permethylated glycans were hydrolysed with HCl/water/acetic acid (0.5:1.5:8, by vol.) at 80oC for 18 h, followed by reduction with 1% NaBD4 in 30mM NaOH and acetylation with acetic anhydride/pyridine (1:1, v/v) at 100 ¿C for 15 min. The partially methylated alditol acetates thus obtained were analyzed by GC-MS. MALDI/TOF-MS analysis For the analyses in negative ion mode, 22,42,62-Trihydroxyacetophenone monohydrate (THAP, 0.5M 2,4,6 THAP in ethanol : 0.1M diammonium hydrogen citrate in water = 2:1 (v/v)) were used as a matrix, whereas, ¿-dihyroxybenzoic acid (DHBA, 20mg/mL solution in 50%methanol: water) were used for the analyses in positive ion mode. For the analysis of intact material, the matrix solution (1ul) was deposited first on the target, then an equal volume of the sample deposited. For the analysis of permethylated materials, the sample solution and an equal amount of matrix were mixed together then 1 uL of the mixture was deposited. All spectra was obtained by using a Microflex LRF (Bruker). MALDI/TOF-MS analysis was performed in the reflector positive or negative ion mode. NSI-MSn analysis Mass analysis was determined by using a LTQ Orbitrap XL mass spectrometer (ThermoFisher) equipped with a nanospray ion source (NSI-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. MSn analysis was performed with ITMS mode with 2.2 isolation width and the collision energy was set at 40. 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 and the collision energy was set at 40. Gas Chromatograph-Mass Spectrometry (GC-MS) The glycosyl linkage analysis was performed on a Hewlett Packard 5890 GC interfaced to a 5970 MSD (mass selective detector, electron impact ionization mode). The separation of the partially methylated alditol acetates (glycosyl linkage analysis) was performed on a 30m EC1 bonded phase fused silica capillary column (Alletech, Deerfield, IL) using a temperature program of 80 oC (2 min)180 oC (20 oC/min)240 oC (4 oC/min). The detector temperature and the inlet temperature were set at 280 oC and 250 oC, respectively.

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：对于 MALDI 分析，按照与之前样品 (PR010510Z-A) 相同的方法分析每个样品的等分试样 (~2ul)。对于糖基连接分析，使用 100ul 样品 B，这些样品已用或未用 laforin 处理。 ，通过 MALDI 和 ESI 分析等份的全甲基化样品（~5%），其余的全甲基化材料被水解。因此，通过 GC-MS 分析得到部分甲基化的糖醇乙酸酯，用于样品分析的详细程序如下所述。 糖基连接分析 1) 全O-甲基化碳水化合物的制备 对样品进行全甲基化以进行糖基键分析，简单地说，将样品溶解在二甲基亚砜中，然后根据Anumula 和Taylor (Anumula 和Taylor, 1992)的方法进行全甲基化，并通过添加水和全-O-来猝灭反应。用二氯甲烷提取甲基化的碳水化合物，通过 MALDI 和 ESI 检查等份的全甲基化，以确认完全的全甲基化。 2) 部分甲基化糖醇乙酸酯的制备 为了测定糖键，由完全全甲基化的聚糖制备部分甲基化的糖醇乙酸酯。简言之，将全甲基化的聚糖用 HCl/水/乙酸（0.5:1.5:8，体积比）在 80℃ 水解 18 小时，然后还原。用 1% NaBD4 的 30mM NaOH 溶液并用乙酸酐/吡啶（1:1， v/v) 在 100¿通过GC-MS分析由此获得的部分甲基化的糖醇乙酸酯。 MALDI/TOF-MS 分析 对于负离子模式的分析，使用 22,42,62-三羟基苯乙酮一水合物（THAP，0.5M 2,4,6 THAP 乙醇溶液：0.1M 柠檬酸氢二铵水溶液 = 2:1 (v/v)）作为矩阵，而 ¿ -二羟基苯甲酸（DHBA，20mg/mL 50%甲醇：水溶液）用于正离子模式分析 对于完整材料的分析，首先将基质溶液（1ul）沉积在目标上，然后将等量的溶液沉积在目标上。为了分析全甲基化材料，将样品溶液和等量的基质混合在一起，然后沉积 1 uL 的混合物。所有光谱均通过使用 Microflex 获得。 LRF (Bruker) 在反射器正离子或负离子模式下进行。 NSI-MSn分析 使用配备纳喷雾离子源（NSI 源）的 LTQ Orbitrap XL 质谱仪（ThermoFisher）进行质量分析，将全甲基化聚糖溶解在 50% 甲醇中的 1mM NaOH 中，并以恒定流速直接注入仪器中。 0.5 ¿ L/min. 以 30 000 分辨率收集完整的 FTMS 谱图，毛细管温度设置为 210oC，MSn 分析采用 ITMS 模式，隔离宽度设置为 2.2。 40。对于总离子图谱（自动 MS/MS 分析），使用 ITMS 模式连续扫描 m/z 范围 300 至 2000 2.8 质量单位窗口与前面的窗口重叠 2 质量单位，碰撞能量设置为 40。 气相色谱-质谱联用仪 (GC-MS) 糖基键分析在连接 5970 MSD（质量选择检测器，电子轰击电离模式）的 Hewlett Packard 5890 GC 上进行。部分甲基化糖醇乙酸酯的分离（糖基键分析）在 30m EC1 键合相融合器上进行。硅胶毛细管柱（Alletech，Deerfield，IL），使用 80 oC 的温度程序(2 分钟) 180 oC (20 oC/min) 240 oC (4 oC/min) 检测器温度和入口温度分别设置为 280 oC 和 250 oC。