O-LINKED OLIGOSACCHARIDE PROFILING OF ONE SAMPLE

一个样品的 O 联寡糖分析

• 批准号: 7956045
• 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/7956045
• 关键词: Acetic Acids; Acetonitriles; Acids; Blood capillaries; Borates; Carbohydrates; Cleaved cell; Complex; Computer Retrieval of Information on Scientific Projects Database; Digestion; Electrophoresis; Formic Acids; Funding; Gases; Gel; Glass; Glycopeptides; Glycoproteins; Grant; Ice; Incubated; Institution; Ions; Link; MALDI-TOF Mass Spectrometry; Maps; Mass Fragmentography; Mass Spectrum Analysis; Methanol; Methods; Nitrogen; Oligosaccharides; Peptides; Phase; Planet Mars; Plant Resins; Polysaccharides; Preparation; Procedures; Proteins; Proteomics; Reaction; Research; Research Personnel; Residual state; Resources; Sampling; Scanning; Sep-Pak C18; Series; Slice; Sodium Hydroxide; Solutions; Source; Stream; Technology; Temperature; Time; Trypsin; Tube; United States National Institutes of Health; Water; ammonium bicarbonate; base; capillary; instrument; ionization; 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. First of all, the glycoprotein sample in IEF gel pieces was digested and extracted from gel pieces following the method of Giorgianni et al. 2003 (Electrophoresis 2003, 24, 253259) and then O-glycans were released from the tryptic digest by b-elimination procedure. The reaction mixture was further desalted, cleaned of borate and released O-glycans were separated from residual peptides by passing though C18 sep-pak cartridge. The O-glycans thus obtained were permethylated and profiled by mass spectrometry. The detailed procedures are shown below. In-gel digestion IEF gel slices were cut into smaller pieces (~3 mm3) and washed with 85% Acetonitrile three times. Dehydrated gels were reswelled with trypsin solution (trypsin in 40 mM Ammonium bicarbonate, AmBic) on ice for 45 min initially, and protein digestion was carried out at 37¿ C overnight. Subsequently, the supernatant was transferred into another tube. Peptides and the glycopeptides were extracted from the gel in series with 20% acetonitrile in 5% formic acid, 50% acetonitrile in 5% formic acid and then 80% acetonitrile in 5% formic acid. The peptides/glycopeptides extracts were dried and combined into one glass tube. O-linked glycan preparation O-linked carbohydrate fractions were cleaved from the tryptic digests by ¿-elimination procedures. Briefly, 500 ¿L of 50 mM Sodium hydroxide (NaOH) containing 19 mg of sodium borohydride were added to the samples and incubated overnight at 45o C. The incubated samples then were neutralized with 10% acetic acid and desalted by passing through a packed column of DOWEXTM resins (50W x 8  100, Sigma Aldrich) and then were lyophilized. Dried samples were cleaned of borate with methanol:acetic acid (9:1) under a stream of nitrogen gas. Then the samples were further passed through a C18 reversed phase cartridge to purify the O-glycans. O-linked glycans were eluted with 5% acetic acid, lyophilized and permethylated based on the method of Anumula and Taylor (Anumula and Taylor, 1992) and profiled by mass spectrometry. Matrix-assisted laser-desorption ionization 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 (LTQ) Mass spectrometric analysis was performed following the method developed at the Complex Carbohydrate 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 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 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。 首先，IEF凝胶片中的糖蛋白样品按照以下方法从凝胶片中消化和提取： Giorgianni等人，2003年（电泳，2003年，24，253259)然后O-聚糖从胰蛋白酶消化物中释放出来， B-消除程序。 将反应混合物进一步脱盐，清除硼酸盐，并释放0-聚糖。 通过C18 sep-pak柱与残留肽分离。 由此获得的0-聚糖是 全甲基化并通过质谱分析。 详细程序如下所示。 凝胶内消化 将IEF凝胶切片切成更小的片（~3 mm 3），并用85%乙腈洗涤三次。 脱水凝胶 最初在冰上用胰蛋白酶溶液（胰蛋白酶在40 mM碳酸氢铵中，AmBic）再溶胀45 min， 蛋白质消化在37 ℃下进行过夜。 随后，将上清液转移到另一个试管中。 肽和糖肽用20%乙腈的5%甲酸溶液、50%乙腈的5%甲酸溶液和50%乙腈的5%甲酸溶液从凝胶中连续提取。 乙腈的5%甲酸溶液，然后80%乙腈的5%甲酸溶液。 肽/糖肽提取物是 干燥并合并到一个玻璃管中。 O-连接聚糖制备 O-连接的碳水化合物部分通过消除程序从胰蛋白酶消化酶中裂解。 简单来说，500 L的50 将含有19 mg硼氢化钠的10 mM氢氧化钠（NaOH）加入样品中并孵育 在45 ° C下过夜。 然后将孵育的样品用10%乙酸中和，并通过 DOWEXTM树脂填充柱（50 W × 8  100，Sigma Aldrich），然后冻干。 干燥的样品 在氮气流下用甲醇：乙酸（9：1）清洗硼酸盐。 然后，样品进一步 通过C18反相柱纯化O-聚糖。 用5%乙酸乙酯洗脱O-连接聚糖。 根据Anumula和Taylor的方法（Anumula和Taylor，1992），将酸冻干并全甲基化， 通过质谱分析。 基质辅助激光解吸电离飞行时间质谱 MALDI/TOF-MS在反射器正离子模式下使用<$-二羟基苯甲酸（DHBA，20 mg/mL溶液）进行 在50%甲醇：水中）作为基质。 通过使用4700蛋白质组学分析仪（Applied Chemistry）获得所有光谱。 Biosystems）。 NanoSpray电离-线性离子阱质谱（LTQ） 按照Complex Carbohydrate Research开发的方法进行质谱分析。 中心（Aoki K，Perlman M，Lim JM，Cantu R，威尔斯L，Tiemeyer M. 2007年3月23日;282（12）：9127-42.）。 质量 通过使用NSI-LTQ/MSn进行分析。 简而言之，将全甲基化聚糖溶解于含50% NaOH的ImM NaOH中。 甲醇，并以0.4 L/min的恒定流速直接注入仪器（LTQ，Thermo Finnigan）。 毛细管温度设定为210 ℃，以正离子模式进行MS分析。 对于总离子绘图，扫描自动MS/MS分析（28碰撞能量），m/z范围500至2000 与前一窗口重叠2个质量单位的连续2.8质量单位窗口。