MONOSACCHARIDE COMPOSITION OF RELEASED N- AND O-LINKED GLYCANS

释放的N-和O-连接聚糖的单糖组合物

• 批准号: 7956036
• 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/7956036
• 关键词: Acetic Acids; Acetonitriles; Acids; Aliquot; Amino Sugars; Analytical Biochemistry; Anions; Borates; Buffers; CD7 gene; Calibration; Carbohydrates; Chromatography; Cleaved cell; Computer Retrieval of Information on Scientific Projects Database; Computer software; Digestion; Dimethyl Sulfoxide; Dryness; Endoglycosidase F; Enzymes; Equation; Excision; Funding; Furuncles; Gases; Glycopeptides; Glycoproteins; Grant; Heating; Hour; Hydrolysis; Ice; Incubated; Individual; Injection of therapeutic agent; Institution; Ions; Isopropanol; Lasers; Link; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Methanol; Methods; Methylene Chloride; Mole the mammal; Monosaccharides; Mus; Needles; New England; Nitrogen; Peptides; Phase; Plant Resins; Polysaccharides; Preparation; Procedures; Proteomics; Protocols documentation; Pump; Reaction; Recombinants; Research; Research Personnel; Resources; Sampling; Sep-Pak C18; Sialic Acids; Sodium Acetate; Solutions; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stainless Steel; Stream; System; Technology; Time; Trifluoroacetic Acid; Trypsin; United States National Institutes of Health; Vial device; Water; ammonium bicarbonate; base; data acquisition; detector; instrument; methyl iodide; programs; sodium borohydride; sugar

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 from glycopeptide The native mouse BChE and recombinant mouse BChE samples were dissolved in ammonium bicarbonate buffer (50 mM, pH 8.4), and denatured immediately by boiling at 100 oC for 5min prior to trypsin digestion at 37 oC for 20 hours respectively. After trypsin digestion, the samples were heated at 100 oC for 5 min to deactivate the enzyme. The samples were passed through a C18 reverse phase cartridge. And then samples were treated with a second enzyme, peptide N-glycosidase F (New England BioLabs) and incubated at 37 oC for 20 hours to release the N-linked glycans. After enzymatic digestion, the samples were passed through a C18 reversed phase cartridge to separate the N-linked glycans and O-linked glycopeptides/peptides. The N-linked glycan fractions of the samples were eluted with 5% acetic acid and then lyophilized. The O-linked glycopeptide/peptide fractions were eluted with 2-propanol and dried under the nitrogen stream. ¿-Elimination, Desalting, Borate removal O-linked carbohydrate fractions were cleaved from the glycopeptides by ¿-elimination procedures. Briefly, 250 ¿L of 50 mM NaOH were added to the sample (400 ¿g) and then checked for pH. Upon determination that the pH was basic, another 250 ¿L of 50 mM NaOH containing 19 mg of sodium borohydride were added to the sample and voltexed and incubated overnight at 45 ¿C. The incubated sample then was 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 sample was cleaned of borate with methanol: acetic acid (9:1) solution under a stream of nitrogen gas. Preparation of the per-O-methylated carbohydrates The lyophilized carbohydrate fraction was dissolved in dimethylsulfoxide and then methylated with NaOH and methyl iodide (Analytical Biochemistry 203, 101-108 (1992)). The reaction was quenched by addition of water, and O- per-methylated carbohydrates were extracted with dichloromethane. The organic phase was concentrated to dryness and then the glycans were passed through a C18 Sep-Pak, eluted with 85 % acetonitrile, dried under a stream of nitrogen, and dissolved in methanol prior to analysis by mass spectrometry. Matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS) Profiling of N-linked and O-linked glycans was performed initially using MALDI/TOF-MS on a 4700 Proteomics analyzer (Applied Biosystems). Permethylated glycans (~1 ¿L) were crystallized on a MALDI plate with 1 ¿L of 2, 3-dihydroxybenzoic acid (DHBA, 20 mg/mL solution in 50 % methanol: water) as matrix. All spectra were acquired in the reflector positive ion mode and averaged spectra of 50 laser shots. Composition analysis by HPAEC Aliquots of the samples were obtained (native, ~70 ¿g each for neutral and amino sugars, and sialic acid analyses; recombinant, ~100 ¿g each for neutral and amino sugars, and sialic acid analyses) for monosaccharide composition analysis of N-linked and O-linked glycans of the two glycoproteins. The released N-and O-linked glycans intended for neutral and amino sugars were hydrolyzed with 400 ¿L of 2 N trifluoroacetic acid (TFA) at 100¿C for 4 h, whereas aliquots for sialic acids were hydrolyzed with 2M acetic acid at 80¿C for 3 h. The hydrolysates were lyophilized, resuspended in H2O, sonicated for 7 min in ice and transferred to an injection vial. A mix of standards for neutral and amino sugars, and for sialic acids with a known number of moles was hydrolyzed in the same manner and at the same time as the samples. Four concentration of standards (0.5, 1.0, 2.0, and 4.0 nmoles per injection) were prepared to establish a calibration equation. The number of moles of each sugar in the sample was quantified by linear interpolation from the calibration equation. The neutral and amino sugars and sialic acids were analyzed by HPAEC using a Dionex DX500 system equipped with a GP40 gradient pump, an ED40 electrochemical detector, and a Thermo-Separation AS3500 autosampler containing a stainless steel needle. The individual neutral and amino sugars were separated by a Dionex CarboPac PA20 (3 x 150 mm) analytical column with an amino trap. The gradient programs used eluents A, degassed nanopure water; B, 200 mM NaOH for the neutral and amino sugars; C, 100 mM NaOH; and D, 1 M sodium acetate in 100 mM NaOH for the sialic acids. Injections were made every 40 minutes for the neutral and amino sugar determinations and every 35 minutes for the sialic acid determinations. All methods were based on protocols described by Hardy and Townsend (Hardy, M. R., and Townsend, R. R., "High-pH anion-exchange chromatography of glycoprotein-derived carbohydrates", 1994, Methods Enzymol. 230: 208-225). Instrument control and data acquisition were accomplished using Dionex PeakNet software, version 5.01.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中央赠款提供的资源 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此，在列出的机构中可能会被压制 对于中心，这不一定是调查员的工厂。 从糖肽中释放N连接的聚糖 将天然小鼠小鼠BCHE样品溶于碳酸氢铵缓冲液（50 mm，pH 8.4），在100 oC中为5分钟，在37 oC的胰蛋白酶消化前20小时，在胰蛋白酶消化后，以100 oc的速度加热。 5分钟与第二个酶，肽N-糖苷酶F（新的英型生物AC），并在37 oC中孵育20小时。 挥之不去 - 淘汰，设计，硼酸盐去除 通过通过„将O连接的碳水化合物级分与糖肽裂解 - 淘汰程序。 l将50 mm的NaOH添加到样品（400 g）中，然后在确定pH时检查pH。将含有19毫克硼氢化钠的50 mm NaOH添加到样品中，并在45€»中孵育过夜。 C.用10％乙酸孵育的样品，并通过theTigh脱盐，将其杂种树脂的填充柱冻结，并用甲醇（9：1）在氮气流中清洗干燥的样品。气体。 甲基化碳水化合物的制备 将冻干的碳水化合物分数溶解在二甲基硫氧化氢中，并用NaOH和甲基碘溶解）试验203，101-108（1992）。和糖通过c18 sep-pak fnogen，而在甲醇中溶于质谱法分析。 基质辅助激光解电电离电离飞行时间质谱（MALDI/TOF-MS） 使用MALDI/TOF-MS ONA 4700蛋白质组学分析仪D Gllycans（〜1？l）在MALDI板上结晶的MALDI板块，对N连锁和链接的聚糖进行了分析。 l 2，3-二羟基苯甲酸（DHBA，20 mg/ml溶液中的50％甲醇：水）作为基质。 HPAEC的组成分析 获得样品的等分试样（用于中性糖和氨基糖，以及唾液酸分析； RAL和氨基糖和唾液酸分析），用于N链接的两种糖蛋白的N-连接和O连接的聚糖的单糖酸组成分析）。 。 释放的N和用于中性和氨基糖的链接的聚糖用400€液化液化。在100°处的2 N三氟乙酸（TFA）的l c持续4小时，而唾液酸的等分试样在80°处用2m乙酸水解C持续3 h。 以相同的方式和四个标准浓度（0.5、1.0、2.0和4.0 nmoles perer注入），将中性和氨基糖的标准和已知摩尔数的唾液酸混合在一起。建立校准方程。 使用配备GP40 A eD40 Cal检测器的DX500系统和HPAEC分析的中性ANO糖和唾液，以及一个含有不符合的钢针的热分离AS3500 AutoSampler。 30毫米）带有梯度的梯度。用于确定中性和氨基糖的分钟，每35分钟r唾液酸确定。衍生的碳水化合物”，方法酶。仪器控制和数据采集使用Dionex Peaknet软件（版本5.01）完成。