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：对于MALDI分析，按照与以前的样品相同的方法（PR010510Z-A）分析了每个样品的等分试样（〜2英尺）。对于糖基链接分析，100 ul的样品B（已接受和没有Laforin处理的样品B）被氯化。 MALDI和ESI分析了苄苄化样品（约5％）的等分试样。将其余的苄苄化材料水解，还原和乙酰化。通过GC-MS对因此获得的部分甲基化的乙醇乙酸乙醇酯。用于样本分析的详细过程如下所述。 糖基链接分析 1）制备每甲基化的碳氢化物 样品被氯化以进行糖基链接分析。简而言之，将样品溶解在二甲基硫氧化氢中，然后基于Anumula和Taylor的方法（Anumula and Taylor，1992），并通过添加水和每种甲基化的碳水化的碳水化合物来淬灭反应，并用二氯甲烷提取反应。 Maldi和ESI检查了苄苄化聚糖的等分试样，以确认完全苄苄丙烯酸化。 2）制备部分甲基化的醛醇加速 为了确定糖连接，从完全苄苄化聚糖中制备了部分甲基化的乙酸乙醇。简而言之，将二甲基化的聚糖用Hcl/水/乙酸水解（0.5：1.5：8，vol。）在80oC下持续18小时，然后在30mm NaOH中用1％Nabd4降低1％Nabd4，并用乙酸酸酐/吡啶乙二醇/吡啶（1：1，V/v）在100€c时以15分钟为单位。通过GC-MS分析了因此获得的部分甲基化的乙醇乙酸。 MALDI/TOF-MS分析 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, ¿ -dihydroxybenzoic acid (DHBA, 20mg/mL在50％甲醇：水中的溶液在正离子模式下用于分析。为了分析完整的材料，首先将矩阵溶液（1英尺）沉积在目标上，然后沉积等同体积的样品。为了分析苄苄化材料，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 LRF（Bruker）获得。 MALDI/TOF-MS分析在反射镜正或负离子模式下进行。 NSI-MSN分析 通过使用配备有纳米喷雾离子源（NSI-Source）的LTQ Orbitrap XL质谱仪（Thermofisher）确定质量分析。将苄苄化聚糖溶解在1mm NaOH中，在50％甲基乙醇中，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。使用2.2隔离宽度进行ITMS模式进行MSN分析，并将碰撞能设置为40。对于总离子映射（自动化的MS/MS分析），M/Z范围，300至2000在成功的2.8个质量单位窗口中扫描了ITMS模式，该2.8质量单位窗口将前面的窗口重叠2个质量单位，并在40张碰撞碰撞。 气相色谱 - 质量光谱法（GC-MS） 糖基链接分析是在连接到5970 MSD（质量选择性检测器，电子撞击电离模式）的惠普（Hewlett Packard）5890 GC上进行的。使用80 OC（2 min）180 OC（20 oC/min）240 OC（4 oc/min）的温度程序（4 oc/min），在30m EC1键合二氧化硅毛细管柱（Alletech，Deerfield，IL）上进行了部分甲基化醛醇（糖基连接分析）的分离。检测器温度和入口温度分别设置为280 OC和250 OC。