O-LINKED OLIGOSACCHARIDE PROFILING MASS SPECTROMETRY

O 联低聚糖分析质谱法

• 批准号: 7956032
• 负责人: Parastoo Azadi
• 金额: $ 0.13万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7956032
• 关键词: 1-Propanol; Acetic Acids; Acetonitriles; Acids; Blood capillaries; Borates; Buffers; Carbohydrates; Cleaved cell; Complex; Computer Retrieval of Information on Scientific Projects Database; Digestion; Dimethyl Sulfoxide; Freeze Drying; Funding; Gases; Glass; Glycopeptides; Grant; Heating; Incubated; Institution; Iodoacetamide; Ions; Lasers; Link; MALDI-TOF Mass Spectrometry; Maps; Mass Fragmentography; Mass Spectrum Analysis; Methanol; Methods; Methylene Chloride; New England; Nitrogen; Oligosaccharides; Peptide N-glycohydrolase F; Peptides; Phase; Planet Mars; Plant Resins; Polysaccharides; Preparation; Procedures; Propanols; Proteomics; Reaction; Research; Research Personnel; Residual state; Resources; Sampling; Scanning; Sep-Pak C18; Series; Solutions; Source; Speed; Stream; Technology; Temperature; Time; Trypsin; Tube; United States National Institutes of Health; Vacuum; Water; ammonium bicarbonate; capillary; instrument; ionization; methyl iodide; sodium borohydride

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The N-glycans were released and removed prior to b-elimination to avoid being mixed with the O-glycans in the latter's analysis. Briefly, glycoroteins were denatured by reduction and carboxyamidomethylation and then treated with Trypsin and PNGase F. Released N-linked glycans were separated from the residual peptides and O-linked glycopeptides through a C18 sep pak cartridge. Subsequently, the O-glycans were cleaved from the O-linked glycopeptides by ¿- elimination procedures. After ¿- elimination, released O-glycans were desalted and cleaned of borate, permethylated and analyzed by MALDI/TOF-MS and NSI-MSn. The procedures are shown in detail below. Release of N-linked glycans The dried sample was dissolved in Ambic buffer (50mM Ammonium Bicarbonate). The sample then was added with 25 mM DTT and incubated for 45 min at 50 for reduction, followed by carboxyamidomethylation with 90mM iodoacetamide, incubated at room temperature in the dark for 45 min. The sample then was digested with the trypsin (37oC, overnight). After digestion, trypsin was inactivated by heating at 100¿ C for 5 min. After cooling to room temperature, the tryptic digest was treated with PNGase F (New England BioLabs) to release the N-glycans. The tryptic/PNGase F digest then was passed through a C18 reversed phase cartridge. The carbohydrate fraction (N-linked glycans) was eluted first with 5% acetic acid and then the O-linked glycopeptides and peptides were eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and 100% iso-propanol each into a microcentrifuge tube. The carbohydrate fraction was dried by lyophilization, whereas the other iso-propanol fractions were dried in a speed vacuum concentrator and then combined into one glass tube. Release of O-linked glycans O-linked carbohydrates were cleaved from the glycopeptides by ¿-elimination procedures. Briefly, 500 ¿L of 50 mM Sodiumhydroxide (NaOH) containing 19 mg of sodium borohydride was added to the sample and incubated overnight at 450C. The incubated sample then was neutralized with 10% acetic acid, desalted by passing through a packed column of DOWEXTM resins (50W x 8  100, Sigma Aldrich) and lyophilized. The dried sample was cleaned of borate with methanol:acetic acid (9:1) under a stream of nitrogen gas. Then the sample was passed through a C18 reversed phase cartridge to separate the O-glycans from the peptides. O-linked glycans were eluted with 5% acetic acid and lyophilized, whereas the peptides were eluted with 100% iso-propanol and dried under a stream of nitrogen gas. Preparation of the per-O-methylated carbohydrates, cleaning up by C18 The dried carbohydrate fraction was dissolved in dimethylsulfoxide and methylated with NaOH and methyl iodide (Anumula and Taylor, 1992). The reaction was quenched by the addition of water and per-O-methylated carbohydrates were extracted with dichloromethane. Per- O-methylated glycans were further cleaned of contaminants. Briefly, the glycans were loaded into a C18 sep pak cartridge and then washed with nanopure water and 15% acetonitrile. The glycans then were eluted with 85% acetonitrile. Purified glycans were dried under a stream of nitrogen gas and dissolved with methanol prior to analysis by mass spectrometry. Matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI/TOF-MS) 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. All spectra were obtained by using a 4700 Proteomics analyzer (Applied Biosystems). NanoSpray ionization-Linear Ion Trap Mass Spectrometry (NSI-LTQ/MSn) Mass spectrometric analysis was performed following the method developed at the Complex Carbohydrates Research Center (Aoki K, Perlman M, Lim JM, Cantu R, Wells L, Tiemeyer M. J Biol Chem. 2007 Mar 23;282(12):9127-42). Mass analysis was determined by using NSI-LTQ/MSn. Briefly, permethylated glycans were dissolved in 1mM NaOH in 50% methanol and infused directly into the instrument (LTQ,Thermo Finnigan) at a constant flow rate of 0.4¿L/min. The capillary temperature was set at 210o C and MS analysis was performed in the positive ion mode. The collision energy was set at 28 for MS/MS fragmentation. For total ion mapping, automated MS/MS analysis (at 28 collision energy), m/z range from 200 to 2000 was scanned in successive 2.8 mass unit windows that overlapped the preceeding window by 2 mass units.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 在B-灭绝之前释放并删除了N-聚糖，以避免在以后的分析中与O-Glycans混合。简而言之，通过还原和羧甲基化将糖蛋白变性，然后用胰蛋白酶和PNGase F处理。释放的N连接聚糖与残留的辣椒分离，并通过C18 sep Pak弹药筒分离出残留的辣椒和O连接的糖肽。随后，O-聚糖通过` - 消除程序都从O连接的糖肽中裂解。 - 进化后，将释放的O-聚糖脱盐并用硼酸盐清洁，苄氯化和通过MALDI/TOF-MS和NSI-MSN进行分析。 该过程在下面详细显示。 释放N连接的聚糖 干燥的样品溶解在Ambic缓冲液（碳酸氢铵50mm）中。然后将样品与25 mm DTT一起添加，并在50下孵育45分钟以减少，然后用90mm碘乙酰氨酰胺羧甲基化，在黑暗的室温下孵育45分钟。然后用胰蛋白酶（37oC，隔夜）消化样品。消化后，通过在100℃加热5分钟来灭活胰蛋白酶。冷却至室温后，将胰蛋白酶消化物用PNGase F（新英格兰生物AB）处理以释放N-聚糖。然后，胰蛋白酶/pNGase f Digest通过C18反向相墨盒传递。首先用5％的乙酸洗脱碳含碳水化合物级分（N-连接的聚糖），然后将O连接的糖肽和肽在5％乙酸中用20％ISO-丙醇串联洗脱，40％ISO丙醇，在5％乙酸中，每种Iso-Propanolopanol op Anecom acty Acety Acetic Acetic Acetic Acetic Acety Acety Acety and Anes Anecimopanolopece and Ane Microcentrif puge to。通过冻干将碳水化合物的馏分干燥，而另一个ISO丙醇馏分则以速度真空浓缩液干燥，然后混合到一个玻璃管中。 释放O连锁聚糖 O连接的碳水化合物通过``省略碳肽''糖肽裂解。简而言之，将含有19毫克硼氢化钠的50 mM钠羟基（NaOH）添加到样品中，并在450°C中孵育过夜。然后，将孵育的样品用10％乙酸中和，通过穿过Dowextm树脂（50W x 8 100，Sigma aldrich）的填充柱并进行冻干而脱盐。干燥的样品用甲醇清洁硼酸盐：乙酸（9：1）在氮气流下。然后，样品通过C18反向相墨盒，将O-Glycans与Petides分开。用5％的乙酸洗脱O连接的糖，并冻干，而用100％ISO丙醇洗脱宠物，并在氮气流下干燥。 制备每甲基化的碳氢化物，由C18清洁 将干碳水化合物馏分溶解在二甲基硫氧化二甲基中，并用NaOH和碘化甲基甲基化（Anumula and Taylor，1992）。通过添加水和每甲基化的碳水化物用二氯甲烷提取反应。将 - 甲基化的甘氨酸进一步清洁污染物。简而言之，将聚糖加载到C18 Sep Pak弹药筒中，然后用纳米水和15％的乙腈洗涤。然后用85％的乙腈洗脱聚糖。在通过质谱法分析之前，将纯化的聚糖干燥在氮气流下，并用甲醇溶解。 基质辅助激光解吸时间飞行时间质谱（MALDI/TOF-MS） 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。所有光谱均通过使用4700个蛋白质组学分析仪（Applied Biosystems）获得。 纳米喷雾电离线性离子陷阱质谱法（NSI-LTQ/MSN） 遵循在复杂碳水化合物研究中心开发的方法（Aoki K，Perlman M，Lim JM，Cantu R，Wells L，Wells L，Tiemeyer M. J BiolChem。20073月23日； 282（12）：9127-42）。使用NSI-LTQ/MSN确定质量分析。简而言之，将苄氨基化的甘氨酸溶解在1mm NaOH中50％甲醇中，并直接以0.4 l/min的恒定流速为0.4。在210O C下设置毛细管温度，并在正离子模式下进行MS分析。对于MS/MS碎片，将碰撞能设置为28。 对于总离子映射，自动化的MS/MS分析（在28碰撞能量时），在成功的2.8个质量单位窗口中扫描了2000至2000的M/Z范围，该窗口与预先窗口重叠的2.8个质量单位窗口被2个质量单元重叠。