N-LINKED OLIGOSACCHARIDE PROFILING (MASS SPECTROMETRY)

N-连接寡糖分析（质谱）

• 批准号: 7956030
• 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/7956030
• 关键词: 1-Propanol; Acetic Acids; Acetonitriles; Acids; Blood capillaries; Carbohydrates; Cleaved cell; Color; Complex; Computer Retrieval of Information on Scientific Projects Database; Coomassie blue; Digestion; Formic Acids; Freeze Drying; Funding; Gel; Glycopeptides; Grant; Heating; Ice; Incubated; Institution; Iodoacetamide; Ions; Link; MALDI-TOF Mass Spectrometry; Maps; Mass Fragmentography; Mass Spectrum Analysis; Methanol; Methods; Oligosaccharides; Peptide N-glycohydrolase F; Peptides; Phosphate Buffer; Planet Mars; Polysaccharides; Preparation; Procedures; Propanols; Proteins; Proteomics; Research; Research Personnel; 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; instrument; ionization; reconstitution; research study; 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. Based on the previous result, we could expect that reducing-end GlcNAc should not be fucosylated with alfa 1-3 and we could expect all N-glycans should be cleaved with PNGase F. Since, PNGase A digestion is skipped in this experiment to avoid losing any sample during the procedure. Other than this, the sample was prepared with same way as previous experiment. The procedures used for this experiment are shown in detail below. <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 140 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. <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 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 210oC and MS analysis was performed in the positive ion mode. For total ion mapping, automated MS/MS analysis (at 30 collision energy), m/z range from 400 to 2000 was scanned in successive 2.8 mass unit windows that overlapped the preceeding window by 2 mass units.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 根据先前的结果，我们可以预期还原端的GlcNAc不应该与alfa1-3岩藻糖基化，我们 可以预期所有的N-糖链都应该被PNGase F切割。因为在这个实验中，PNGase A的消化被跳过了，以 避免在操作过程中丢失任何样品。 除此之外，样品的制备方法与之前的实验相同。 下面将详细说明用于该实验的步骤。 &lt；凝胶内消化&gt； 考马斯蓝染色的凝胶切片切成更小的碎片(~1mm3)，交替脱色40 mm 碳酸氢铵(AmBic)和100%乙腈，直到颜色变得透明。死因凝胶剂在10个月内重新溶出 MM DTT在40 mM Ambic中，55℃下放置1小时。DTT溶液与55 mM碘乙酰胺(IAM)交换， 在黑暗中孵化45分钟。孵化后用40 mM AmBic和100%交替洗涤 乙腈两次。脱水凝胶用胰酶溶液(140 mM Ambic中的胰酶)在冰上重新膨胀45min 最初，蛋白质消化在37℃下进行过夜。将上清液转移到另一试管中。 用20%乙腈-5%甲酸-50%乙腈从凝胶中提取多肽和糖肽。 乙腈用5%的甲酸，然后80%的乙腈用5%的甲酸。将样品溶液干燥并 合并成一根管子。 &lt；葡聚糖制剂&gt； 提取的胰酶消化液通过C18 Sep-pak色谱柱，用5%的冰醋酸洗涤以去除 污染物(盐、十二烷基硫酸钠等)。多肽和糖肽用20%异丙醇在5%醋酸中串联洗脱 酸，40%异丙醇在5%醋酸和100%异丙醇中，在高速真空浓缩器中干燥。干的 将样品合并，然后用50 mM磷酸二氢钠缓冲液(pH 7.5)重新组合，并在100℃加热 5分钟灭活胰酶。胰酶消化液与PNGase F在37℃下孵育一夜，以释放N-糖链。 消化后，样品通过C18 Sep-pak色谱柱，碳水化合物部分用 5%冰醋酸，冷冻干燥。根据该方法对释放的N-连接低聚糖进行全甲基化 阿努穆拉和泰勒(Anuula and Taylor，1992)，并通过质谱学进行了分析。 基质辅助激光解吸电离飞行时间质谱仪(MALDI/TOF-MS) MALDI/TOF-MS在反射镜正离子模式下进行，使用2-二羟基苯甲酸(DHBA，20 mg/mL溶液 在50%甲醇：水中)作为基质。使用4700蛋白质组学分析仪(已应用)获得所有光谱 生物系统)。 纳米螺旋电离-线性离子陷阱质谱仪(LTQ)和GT； 根据复合碳水化合物研究开发的方法进行质谱分析 中心(青木K，Perlman M，Lim JM，坎图R，威尔斯·L，蒂迈耶·M·生物化学。2007年3月23日；282(12)：9127-42。质量 采用NSI-LTQ/MSN软件进行分析。简而言之，全甲基化的葡聚糖在50%的1 mM氢氧化钠中溶解。 甲醇，直接进入仪器(LTQ，Thermo Finnigan)，恒定流速为0.4？L/分钟。这个 毛细管温度210oC，正离子模式下进行MS分析。 对于总离子图谱，自动MS/MS分析(碰撞能量为30)，扫描范围从400到2000的m/z 与前一个窗口重叠2个质量单位的连续2.8个质量单位窗。