COMPOSITION ANALYSIS OF 17 EGG WHITE SAMPLES N-GLYCANS BY HPAEC

通过 HPAEC 对 17 个蛋清样品 N-聚糖进行成分分析

基本信息

批准号：
8170802
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
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-06-01 至 2011-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8170802
关键词：
Acetic Acids Acetone Aliquot Amino Sugars Anions Buffers CD7 gene Calibration Carbohydrates Chloroform Chromatography Computer Retrieval of Information on Scientific Projects Database Computer software Egg White Enzymes Equation Excision Funding Gases Glass Glycopeptides Glycoproteins Grant Heating Hour Hydrolysis Ice Incubated Individual Injection of therapeutic agent Institution Isopropanol Link Lipids Methanol Methods Mole the mammal Monosaccharides Needles Nitrogen Peptide N-glycohydrolase F Peptides Phosphate Buffer Polysaccharides Precipitation Proteins Protocols documentation Pump Research Research Personnel Resources Sampling Sep-Pak C18 Series Sialic Acids Sodium Acetate Source Stainless Steel Stream System Temperature Time Trifluoroacetic Acid Trypsin Tube United States National Institutes of Health Vial device Water ammonium bicarbonate base chymotrypsin data acquisition detector instrument polypeptide programs sodium phosphate sugar

项目摘要

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. Lipid extraction and protein precipitation All samples were transferred from their original containers into screw-cap glass tubes. Lipids were extracted from the samples three times with chloroform:methanol:water (4:8:3). After lipid extraction, proteins were precipitated with acetone:water (4:1) in ice with the concomitant removal of free sugars and contaminants. Release of N-linked glycans An aliquot (to provide ~1.5 mg) of each sample was lyophilized and was dissolved in ammonium bicarbonate buffer (50 mM, pH 8.4). The samples were placed in a heating block at 100oC for 5 min to denature protein. After cooling to room temperature, the samples were treated with trypsin and chymotrypsin and incubated at 37oC overnight. Each of the tryptic-chymotryptic digests was passed through a C18 sep pak cartridge, cleaned with 5% acetic acid, and glycopeptides/peptides were eluted subsequently in series with 20% isopropanol in 5% acetic acid, 40% isopropanol in 5% acetic acid and 100% isopropanol. The eluates were dried initially under a stream of nitrogen gas and eventually lyophilized. The dried eluates were dissolved with sodium phosphate buffer, treated with PNGase F and incubated at 37oC for 18 hours to release N-linked glycans from the polypeptide chains. After incubation, the enzyme (PNGase F) digests were passed through C18 sep pak cartridges and N-linked glycans fractions were eluted with 5% acetic acid and lyophilized. Monosaccharide composition analysis by HPAEC Each of the N-linked glycans fractions was divided into two aliquots: one for neutral and amino sugars analysis and the other aliquot for sialic acid analysis. The aliquots intended for neutral and amino sugars analysis were hydrolyzed with 2 N trifluoroacetic acid at 100oC for 4 hours and those for sialic acid analysis were hydrolyzed with 2 M acetic acid at 80oC for 3 hours. The hydrolysates were then lyophilized, redissolved 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 sample. Four concentration of standard mix (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, and sialic acids 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; and C, 100 mM NaOH for the neutral and amino sugars; and C and D, 1 M sodium acetate in 100 mM NaOH for sialic acids. Injections were made every 40 minutes for neutral and amino sugar determinations and every 35 minutes for 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.

该子项目是利用该技术的众多研究子项目之一资源由 NIH/NCRR 资助的中心拨款提供。子项目和研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金，因此可以在其他 CRISP 条目中表示。列出的机构是中心，不一定是研究者的机构。脂质提取和蛋白质沉淀所有样品均从其原始容器转移至螺旋盖玻璃管中。用氯仿:甲醇:水(4:8:3)从样品中提取脂质3次。脂质提取后，用丙酮:水 (4:1) 在冰中沉淀蛋白质，同时去除游离糖和污染物。 N-连接聚糖的释放将每个样品的等分试样（约 1.5 mg）冻干并溶解在碳酸氢铵缓冲液（50 mM，pH 8.4）中。将样品置于 100°C 的加热块中 5 分钟以使蛋白质变性。冷却至室温后，将样品用胰蛋白酶和胰凝乳蛋白酶处理，并在37℃下孵育过夜。每个胰蛋白酶-胰凝乳蛋白酶消化物都通过 C18 sep pak 柱，用 5% 乙酸清洗，随后用 20% 异丙醇的 5% 乙酸溶液、40% 异丙醇的 5% 乙酸溶液连续洗脱糖肽/肽和 100% 异丙醇。洗脱液最初在氮气流下干燥并最终冻干。将干燥的洗脱液用磷酸钠缓冲液溶解，用 PNGase F 处理并在 37°C 下孵育 18 小时，以从多肽链中释放 N-连接聚糖。孵育后，酶 (PNGase F) 消化物通过 C18 sep pak 柱，N-连接聚糖组分用 5% 乙酸洗脱并冻干。 HPAEC 的单糖组成分析每个 N 连接聚糖级分均分为两等份：一份用于中性糖和氨基糖分析，另一份用于唾液酸分析。用于中性糖和氨基糖分析的等分试样用 2 N 三氟乙酸在 100°C 下水解 4 小时，用于唾液酸分析的等分试样用 2 M 乙酸在 80°C 下水解 3 小时。然后将水解产物冻干，重新溶解在水中，在冰中超声处理 7 分钟，然后转移至注射瓶中。以与样品相同的方式和同时水解中性糖和氨基糖以及已知摩尔数唾液酸的标准混合物。制备四种浓度的标准混合物（每次注射 0.5、1.0、2.0 和 4.0 纳摩尔）以建立校准方程。通过校准方程的线性插值来量化样品中每种糖的摩尔数。使用配备 GP40 梯度泵、ED40 电化学检测器和包含不锈钢针的 Thermo-Separation AS3500 自动进样器的 Dionex DX500 系统，通过 HPAEC 分析中性糖、氨基糖和唾液酸。通过带有氨基捕集器的 Dionex CarboPac PA20 (3 x 150 mm) 分析柱分离各个中性糖、氨基糖以及唾液酸。梯度程序使用洗脱液A，脱气纳米纯水； B、200 mM NaOH； C，100 mM NaOH 用于中性糖和氨基糖； C 和 D，1 M 乙酸钠的 100 mM NaOH 溶液用于唾液酸。每 40 分钟注射一次以测定中性糖和氨基糖，每 35 分钟注射一次以测定唾液酸。所有方法均基于Hardy和Townsend描述的方案（Hardy，M.R.和Townsend，R.R.，“糖蛋白衍生碳水化合物的高pH阴离子交换层析”，1994，Methods Enzymol.230：208-225）。使用 Dionex PeakNet 软件 5.01 版完成仪器控制和数据采集。