N-LINKED OLIGOSACCHARIDE PROFILING

N-连接寡糖分析

• 批准号: 8363108
• 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/8363108
• 关键词: 1-Propanol; Acetic Acids; Acetonitriles; Acids; Blood capillaries; Carbohydrates; Color; Coomassie blue; Digestion; Formic Acids; Freeze Drying; Funding; Gel; Glycopeptides; Grant; Heating; Ice; Incubated; Iodoacetamide; Ions; Link; MALDI-TOF Mass Spectrometry; Maps; Mass Spectrum Analysis; Methanol; Methods; National Center for Research Resources; Oligosaccharides; Peptide N-glycohydrolase F; Peptides; Phosphate Buffer; Polysaccharides; Preparation; Principal Investigator; Propanols; Proteins; Research; Research Infrastructure; Resolution; Resources; Salts; Sampling; Scanning; Sep-Pak C18; Series; Slice; Solutions; Source; Speed; Staining method; Stains; Technology; Temperature; Trypsin; Tube; United States National Institutes of Health; Vacuum; Water; ammonium bicarbonate; base; capillary; cost; instrument; ion source; ionization; 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. 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. <In-gel digestion> Coomassie blue-stained gel slices were cut into smaller pieces (~1 mm3) and destained alternately with 40mM Ammonium bicarbonate (AmBic) and 100% acetonitrile until the color turned clear. Destained gel was reswelled in 10 mM DTT in 40mM Ambic at 55¿ C for 1 hr. The DTT solution was exchanged with 55mM Iodoacetamide (IAM) and incubated in the dark for 45 min. Incubation was followed by washing alternately with 40mM AmBic and 100% acetonitrile twice. Dehydrated gel was reswelled with trypsin solution (trypsin in 40 mM Ambic) on ice for 45 min initially, and protein digestion was carried out at 37¿ C overnight. 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 sample solutions were dried and combined into one tube. <Glycan preparation> Extracted tryptic digest was passed through a C18 sep-pak cartridge and washed with 5% acetic acid to remove contaminants (salts, SDS, etc.). Peptides and glycopeptides were eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and 100% iso-propanol and dried in a speed vacuum concentrator. The dried samples were combined and then reconstituted with 50 mM sodium phosphate buffer (pH 7.5) and heated at 100¿ C for 5 min to inactivate trypsin. The tryptic digest was incubated with PNGase F at 37¿ C overnight to release N-glycans. After digestion, the sample was passed through a C18 sep-pak 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 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 Microflex LRF (Bruker). NSI(nanospray ionization)-MSn analysis was performed 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 0.5 ¿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. For total ion mapping (automated MS/MS analysis), m/z range, 800 to 2000 was scanned with ITMS mode in successive 2.8 mass unit windows that overlapped the preceding window by 2 mass units.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 <In-gel digestion> 将考马斯亮蓝染色的凝胶切片切成更小的片（约1 mm 3），并用40 mM碳酸氢铵（AmBic）和100%乙腈交替脱色，直至颜色变澄清。 将脱色的凝胶在55 ℃下在40 mM Ambic中的10 mM DTT中再溶胀1小时。将DTT溶液与55 mM碘乙酰胺（IAM）交换，并在黑暗中孵育45分钟。孵育后用40 mM AmBic和100%乙腈交替洗涤两次。 脱水凝胶最初在冰上用胰蛋白酶溶液（40 mM Ambic中的胰蛋白酶）再溶胀45分钟，并在37 ℃下进行蛋白质消化过夜。将上清液转移至另一管中。 用20%乙腈的5%甲酸溶液、50%乙腈的5%甲酸溶液、然后80%乙腈的5%甲酸溶液从凝胶中连续提取肽和糖肽。 将样品溶液干燥并合并到一个试管中。 <Glycan preparation> 将提取的胰蛋白酶消化物通过C18 sep-pak柱，并用5%乙酸洗涤以除去污染物（盐、SDS等）。 用20%异丙醇的5%乙酸溶液、40%异丙醇的5%乙酸溶液和100%异丙醇连续洗脱肽和糖肽，并在高速真空浓缩器中干燥。 合并干燥的样品，然后用50 mM磷酸钠缓冲液（pH 7.5）复溶，并在100 ℃下加热5 min至胰蛋白酶。 将胰蛋白酶消化物与PNGase F在37 ℃下孵育过夜，以释放N-聚糖。 消化后，使样品通过C18 sep-pak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。 <Mass spectrometry> 采用反射器正离子模式，以20 mg/mL的DHBA（50%甲醇：水）为基质，进行MALDI/TOF-MS。 通过使用Microflex LRF（Bruker）获得所有光谱。 通过使用配备有纳米喷雾离子源的LTQ Orbitrap XL质谱仪（ThermoFisher）进行NSI（纳米喷雾电离）-MSn分析。 将全甲基化聚糖溶于1 mM NaOH的50%甲醇溶液中，并以0.5 μ L/min的恒定流速直接注入仪器。以30000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（自动MS/MS分析），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800至2000。