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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 中心，不一定是研究者的机构。 方法： 样品似乎含有去污剂（Triton、Tween 等...），可能会干扰样品分析，因此在通过质谱分析样品之前，用丙酮清洗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 将丙酮加入到干燥的样品中，将样品溶液放置在冰上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。将样品洗涤3次。 N-连接聚糖的释放 将干燥的样品溶解在胰蛋白酶缓冲液（0.1M Tris-HCl，pH 8.2，含有 0.01M CaCl2）中，然后在 100° 下加热 5 分钟使样品变性。 C.冷却后，用胰蛋白酶消化样品（37℃，过夜）。胰蛋白酶消化后，将样品加热至100°C。 C 5 分钟使胰蛋白酶失活。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 清洁 C18 sep-pak 柱中吸附的样品。 ％乙酸以去除任何可能的污染物（盐等），然后用 20％ 的乙酸连续洗脱。将 5% 乙酸中的异丙醇、5% 乙酸中的 40% 异丙醇和 100% 异丙醇分别放入微量离心管中，然后将丙醇级分合并在一根管中。用 50mM 钠重新溶解样品。磷酸盐缓冲液（pH 7.5），然后使用 PNGase F（新英格兰生物实验室）释放 N-聚糖。消化后，样品通过 C18 反相柱，用 5% 乙酸洗脱碳水化合物部分，并根据 Anumula 和 Taylor 的方法通过冻干干燥释放的 N-连接寡糖（Anumula 和 Taylor，1992）。并通过质谱分析进行分析。 质谱分析 MALDI/TOF-MS 分析使用 ¿ 在反射器正离子模式下进行-二羟基苯甲酸（DHBA，20mg/mL 50%甲醇：水溶液）作为基质。所有光谱均通过使用 4700 蛋白质组分析仪（Applied Biosystems）获得。 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 K, Perlman M, Lim JM, Cantu R, Wells L, Tiemeyer M. J Biol Chem. 2007 Mar 23;282(12):9127- 42.) 使用配备纳喷雾的 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 0.4 ¿ 的恒定流速直接注入仪器中。 L/分钟以 60 000 分辨率收集完整的 FTMS 谱图，毛细管温度设置为 210oC，并在正离子模式下进行 MS 分析。 对于总离子图谱，使用 ITMS 模式在连续 2.8 质量单位窗口中扫描自动 MS/MS 分析（在 28 碰撞能量下），m/z 范围为 500 至 2000，该窗口与前面的窗口重叠 2 质量单位。