N-LINKED OLIGOSACCHARIDE PROFILING OF ONE SAMPLE

一个样品的 N 联寡糖分析

• 批准号: 8170789
• 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: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170789
• 关键词: 1-Propanol; Acetic Acids; Acetone; Acids; Blood capillaries; Buffers; Carbohydrates; Complex; Computer Retrieval of Information on Scientific Projects Database; Detergents; Digestion; Freeze Drying; Funding; Glycopeptides; Grant; Heating; Ice; Institution; Ions; Link; MALDI-TOF Mass Spectrometry; Maps; Mass Spectrum Analysis; Methanol; Methods; New England; Oligosaccharides; Peptide N-glycohydrolase F; Peptides; Phase; Phosphate Buffer; Planet Mars; Polysaccharides; Precipitation; Procedures; Propanols; Proteins; Proteomics; Research; Research Personnel; Resolution; Resources; Salts; Sampling; Scanning; Sep-Pak C18; Series; Solutions; Source; Speed; Temperature; Time; Triton; Trypsin; Tube; Tweens; United States National Institutes of Health; Water; base; capillary; instrument; ion source; mass spectrometer; reconstitution; sodium phosphate

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. Methods: The sample seemed to contain detergents (Triton, Tween and so on...), which may interfere with the sample analysis, so the sample was washed with acetone to make sure to remove all detergents before we analyze the sample by mass spectrometry. The detailed procedures used for your sample are shown below. Acetone precipitation Acetone was added to the dried sample. The sample solution was placed on ice for 15 minutes and then spun at maximum speed in a refrigerated microcentrifuge for 15 minutes to pellet the protein. The supernatant was removed. The sample was washed three times. Release of N-linked glycans The dried sample was dissolved in Trypsin buffer (0.1M Tris-HCl, pH 8.2 containing 0.01M CaCl2). The sample then was denatured by heating for 5 minutes at 100¿C. After cooling, the sample was digested with the trypsin (37oC, overnight). After tryptic digestion, the sample was heated at 100¿ C for 5 minutes to de-activate the trypsin. After cooling to room temperature, the sample was applied to a C18 sep-pak cartridge. Before elution of glycopeptides and peptides, the sample adsorbed in the C18 sep-pak cartridge was cleaned with 5% acetic acid to remove any possible contaminants (salts, etc.). Peptides and glycopeptides then 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 propanol fractions were dried and then combined in one tube. The sample was reconstituted with 50mM sodium phosphate buffer (pH 7.5) and then the N-glycans were released using PNGase F (New England BioLabs). After digestion, the sample was passed through a C18 reversed phase cartridge and the carbohydrate fraction was eluted with 5% acetic acid and dried by lyophilization. Released N-linked oligosaccharides were permethylated based on the method of Anumula and Taylor (Anumula and Taylor, 1992) and profiled by mass spectrometry. Mass spectrometry MALDI/TOF-MS analysis 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). NSI-MSn 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 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 0.4 ¿L/ min. A full FTMS spectrum was collected at 60 000 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 (at 28 collision energy), m/z range, 500 to 2000 was scanned with ITMS mode in successive 2.8 mass unit windows that overlapped the preceding window by 2 mass units.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 研究方法： 样品似乎含有洗涤剂（Triton、Tween等），这可能会干扰样品分析，因此在我们通过质谱法分析样品之前，用丙酮洗涤样品以确保去除所有去污剂。 用于您的示例的详细程序如下所示。 丙酮沉淀 将丙酮加入干燥样品中。 将样品溶液置于冰上15分钟，然后在冷冻微量离心机中以最大速度旋转15分钟以沉淀蛋白质。 除去上清液。 将样品洗涤三次。 N-连接聚糖的释放 将干燥的样品溶解于胰蛋白酶缓冲液（0.1M Tris-HCl，pH 8.2，含有0.01M CaCl 2）中。 然后通过在100 ℃下加热5分钟使样品变性。 冷却后，用胰蛋白酶消化样品（37 ℃，过夜）。 胰蛋白酶消化后，将样品在100 ℃下加热5分钟以使胰蛋白酶失活。 冷却至室温后，将样品上样至C18 sep-pak柱。 在洗脱糖肽和肽之前，用5%乙酸清洁吸附在C18 sep-pak柱中的样品，以去除任何可能的污染物（盐等）。 然后将肽和糖肽用20%异丙醇的5%乙酸溶液、40%异丙醇的5%乙酸溶液和100%异丙醇依次洗脱到微量离心管中。 将丙醇级分干燥，然后在一个管中合并。 用50 mM磷酸钠缓冲液（pH 7.5）复溶样品，然后使用PNGase F（新英格兰BioLabs）释放N-聚糖。 消化后，使样品通过C18反相柱，用5%乙酸洗脱碳水化合物级分，并通过冻干干燥。 根据Anumula和Taylor的方法（Anumula和Taylor，1992）对释放的N-连接寡糖进行全甲基化，并通过质谱分析。 质谱 以反射器正离子模式进行MALDI/TOF-MS分析，使用<$-二羟基苯甲酸（DHBA，20 mg/mL的50%甲醇：水溶液）作为基质。 通过使用4700蛋白质组学分析仪（Applied Biosystems）获得所有光谱。 按照复杂碳水化合物研究中心开发的方法进行NSI-MSn分析（Aoki K，Perlman M，Lim JM，Cantu R，威尔斯L，Tiemeyer M. 2007年3月23日;282（12）：9127-42.）。 通过使用配备有纳米喷雾离子源的LTQ Orbitrap XL质谱仪（ThermoFisher）测定质量分析。 将全甲基化聚糖溶于1 mM NaOH的50%甲醇溶液中，并以0.4 μ L/min的恒定流速直接注入仪器中。以60000分辨率收集完整的FTMS光谱。 毛细管温度设定为210 ℃，以正离子模式进行MS分析。 对于总离子图谱，在与前一窗口重叠2个质量单位的连续2.8质量单位窗口中，用ITMS模式扫描m/z范围为500至2000的自动MS/MS分析（在28碰撞能量下）。