O-LINKED OLIGOSACCHARIDE PROFILING OF TWO SAMPLES

两个样品的 O 联寡糖分析

• 批准号: 7722684
• 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/7722684
• 关键词: 1-Propanol; Acetic Acid; Acetic Acids; Acetonitriles; Acids; Blood capillaries; Borates; Buffers; Carbohydrates; Cleaved cell; Complex; Computer Retrieval of Information on Scientific Projects Database; Digestion; Dimethyl Sulfoxide; Freeze Drying; Funding; Gases; Glycopeptides; Glycoproteins; Grant; Heating; Incubated; Institution; Ions; Lasers; Link; MALDI-TOF Mass Spectrometry; Maps; Mass Fragmentography; Mass Spectrum Analysis; Methanol; Methods; Methylene Chloride; New England; Nitrogen; Oligosaccharides; Peptide N-glycohydrolase F; Peptides; Phase; Planet Mars; Plant Resins; Polysaccharides; Preparation; Procedures; Propanols; Proteomics; Range; Rate; Reaction; Research; Research Personnel; Resources; Sampling; Scanning; Sep-Pak C18; Series; Solutions; Source; Speed; Stream; Temperature; Time; Trypsin; Tube; United States National Institutes of Health; Vacuum; Water; capillary; instrument; ionization; methyl iodide; 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. Release of N-linked glycans The dried samples were dissolved in Trypsin buffer (0.1M Tris-HCl, pH 8.2 containing 0.01M CaCl2). The samples then were denatured by heating for 5 minutes at 100¿C. After cooling, the samples were digested with the trypsin (37oC, overnight). After tryptic digestion, the samples were heated at 100¿ C for 5 minutes to de-activate the trypsin. After cooling to room temperature, the N-glycans were treated with PNGase F (New England BioLabs) to release the N-glycans. After digestion, the samples were passed through a C18 reversed phase cartridge. The carbohydrate fraction (N-linked glycans) was first eluted with 5% acetic acid and then the O-linked glycopeptides and peptides 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 carbohydrate fraction was dried by lyophilization, whereas the other propanol fractions were dried in a speed vacuum concentrator and then combined into one tube. Release of O-linked glycans O-linked carbohydrate fractions were cleaved from the glycoprotein by ¿-elimination procedures. Briefly, 500 ¿L of 50 mM Sodiumhydroxide (NaOH) containing 19 mg of sodium borohydride were added to the samples and incubated overnight at 450C. 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 passed through a C18 reversed phase cartridge to purify the O-glycans. The carbohydrate fractions (O-linked glycans) were first eluted with 5% acetic acid and then the peptides were eluted with 100% iso-propanol. The carbohydrate fractions were dried by lyophilization, whereas the propanol fractions were dried under a stream of nitrogen gas. Preparation of the per-O-methylated carbohydrates, cleaning up by C18 The carbohydrate fraction was dissolved in dimethylsulfoxide and then methylated with NaOH and methyl iodide (Ciucanu and Kerek, 1984). The reaction was quenched by addition of water and per-O-methylated carbohydrates were extracted with dichloromethane. Per- O-methylated glycans were further cleaned of contaminants. Briefly, the glycans were loaded into a C18 sep pak cartridge and then washed with nanopure water and 15% acetonitrile. The glycans then were eluted with 85% acetonitrile. Purified glycans were dried under a stream of nitrogen gas and were dissolved with methanol prior to analysis by mass spectrometry. Matrix-assisted laser-desorption 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 (NSI-LTQ/MSn) 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 210o C and MS analysis was performed in the positive ion mode. The collision energy was set at 28 for MS/MS fragmentation. 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.

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。 N-连接聚糖的释放 将干燥的样品溶解于胰蛋白酶缓冲液（0.1M Tris-HCl，pH 8.2，含有0.01M CaCl 2）中。然后通过在100 ℃下加热5分钟使样品变性。 冷却后，用胰蛋白酶消化样品（37 ℃，过夜）。 胰蛋白酶消化后，将样品在100 ℃下加热5分钟以使胰蛋白酶失活。 冷却至室温后，用PNGase F（新英格兰BioLabs）处理N-聚糖以释放N-聚糖。 消化后，将样品通过C18反相柱。 首先用5%乙酸洗脱碳水化合物级分（N-连接聚糖），然后用20%异丙醇/5%乙酸、40%异丙醇/5%乙酸和100%异丙醇将O-连接糖肽和肽依次洗脱至微量离心管中。 通过冻干干燥碳水化合物级分，而在高速真空浓缩器中干燥其它丙醇级分，然后合并到一个管中。 O-连接聚糖的释放 O-连接的碳水化合物部分通过消除程序从糖蛋白中裂解。 简言之，向样品中加入500 μ L含有19 mg硼氢化钠的50 mM氢氧化钠（NaOH），并在45 ℃下孵育过夜。 然后将温育的样品用10%乙酸中和，并通过DOWEXTM树脂的填充柱（50 W × 8）脱盐  100，Sigma Aldrich），然后冻干。 在氮气流下用甲醇：乙酸（9：1）清洗干燥样品的硼酸盐。 然后将样品通过C18反相柱以纯化O-聚糖。 首先用5%乙酸洗脱碳水化合物级分（O-连接聚糖），然后用100%异丙醇洗脱肽。 碳水化合物部分通过冻干干燥，而丙醇部分在氮气流下干燥。 制备全-O-甲基化的碳水化合物，通过C18净化 将碳水化合物部分溶于二甲亚砜中，然后用NaOH和碘甲烷甲基化（Ciucanu和Kerek，1984）。 通过加入水淬灭反应，用二氯甲烷萃取全-O-甲基化的碳水化合物。 进一步清除全-0-甲基化聚糖的污染物。 简言之，将聚糖加载到C18 sep pak柱中，然后用纳米纯水和15%乙腈洗涤。 然后用85%乙腈洗脱聚糖。 在氮气流下干燥纯化的聚糖，并用甲醇溶解，然后通过质谱法进行分析。 基质辅助激光解吸飞行时间质谱（MALDI/TOF-MS） 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4700蛋白质组学分析仪（Applied Biosystems）获得所有光谱。 NanoSpray电离-线性离子阱质谱（NSI-LTQ/MSn） 按照复杂碳水化合物研究中心开发的方法进行质谱分析（Aoki K，Perlman M，Lim JM，Cantu R，威尔斯L，Tiemeyer M. 2007年3月23日;282（12）：9127-42）。通过使用NSI-LTQ/MSn确定质量分析。 简言之，将全甲基化聚糖溶于1 mM NaOH的50%甲醇溶液中，并以0.4 μ 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。 MS/MS裂解的碰撞能量设定为28。 对于总离子图谱，自动MS/MS分析（在28个碰撞能量下），在连续的2.8个质量单位窗口中扫描500至2000的m/z范围，该窗口与前一窗口重叠2个质量单位。