N-LINKED OLIGOSACCHARIDE PROFILING OF ONE SAMPLE

一个样品的 N 联寡糖分析

• 批准号: 7722688
• 负责人: Parastoo Azadi
• 金额: $ 0.02万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-08 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7722688
• 关键词: 1-Propanol; Acetic Acid; Acetic Acids; Acetonitriles; Acids; Blood capillaries; Carbohydrates; Color; Complex; Computer Retrieval of Information on Scientific Projects Database; 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; Propanols; Proteins; Proteomics; Range; Rate; Research; Research Personnel; Resources; Salts; Sampling; Scanning; Sep-Pak C18; Series; Slice; Solutions; Source; Speed; Staining method; Stains; Temperature; Trypsin; Tube; United States National Institutes of Health; Vacuum; Water; ammonium bicarbonate; capillary; formic acid; instrument; ionization; 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. <In-gel digestion> Coomassie 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 according to the method of Ciucanu and Kerek (1984) 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 28 collision energy), m/z range from 500 to 2000 was scanned in successive 2.8 mass unit windows that overlapped the preceeding window by 2 mass units.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 <凝胶消化> 切成较小的碎片（〜1 mm3），用40mm碳酸氢铵（Ambic）和100％的乙腈切成薄片，直到颜色变为透明，将其切成较小的碎片（〜1 mm3）。在40毫米Ambic的55°C中，DeStained Gel在10毫米DTT中被重新温度持续1小时。将DTT溶液与55mm的碘乙酰胺（IAM）交换，并在黑暗中孵育45分钟。孵育之后，用40mm的Ambic和100％乙腈洗涤两次。脱水的凝胶用胰蛋白酶溶液（140毫米Ambic中的胰蛋白酶溶液）在冰上持续45分钟，然后在37°C下进行蛋白质消化过夜。将上清液转移到另一个管中。从5％甲酸中的5％甲酸，5％甲酸中的50％乙腈中从凝胶中提取肽和糖肽，然后在5％的甲酸中提取50％的乙腈，然后在5％的甲酸中提取80％乙腈。将样品溶液干燥并组合成一个管。 <Glycan准备> 提取的胰蛋白酶消化物通过C18 sep-pak墨盒，并用5％乙酸洗涤以去除污染物（盐，SDS等）。在5％乙酸中以20％ISO丙醇为序列的肽和糖磷酸，在5％乙酸中40％ISO-丙醇和100％ISO丙醇，并在速度真空浓缩剂中干燥。将干燥的样品合并，然后用50 mM磷酸钠缓冲液（pH 7.5）重构，并在100°C加热5分钟以灭活胰蛋白酶。将胰蛋白酶消化物与37 c的PNGASE F一起孵育，以释放N-聚糖。消化后，样品通过C18 sep-pak弹药筒，用5％乙酸洗脱碳水化合物馏分，并通过冻干干燥。根据Ciucanu和Kerek（1984）的方法将释放的N连接寡糖泛氧化物化，并通过质谱法进行了介绍。 <基质辅助激光解析电离飞行时间质谱（MALDI/TOF-MS）> MALDI/TOF -MS在反射器正离子模式下使用�-二羟基苯甲酸（DHBA，20mg/ml溶液中的50％甲烷：水）作为基质进行。所有光谱均通过使用4700个蛋白质组学分析仪（Applied Biosystems）获得。 <纳米喷雾电离线性离子陷阱质谱法（LTQ）> 遵循在复杂碳水化合物研究中心开发的方法（Aoki K，Perlman M，Lim JM，Cantu R，Wells L，Wells L，Tiemeyer M. J BiolChem。20073月23日； 282（12）：9127-42。）。使用NSI-LTQ/MSN确定质量分析。简而言之，将苄氨基化的甘氨酸溶解在1mm NaOH中，在50％甲醇中，并直接注入仪器（LTQ，Thermo Finnigan）中，恒定流速为0.4。在210oC处设置毛细管温度，并在正离子模式下进行MS分析。 对于总离子映射，自动化的MS/MS分析（在28碰撞能量时），在成功的2.8个质量单位窗口中扫描了500至2000的M/Z范围，该窗口与前面的窗口重叠了2个质量单位。