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

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。 脂肪提取和蛋白质沉淀 所有样品都从原来的容器中转移到螺帽玻璃管中。样品用氯仿：甲醇：水(4：8：3)提取3次。提取脂肪后，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-连接葡聚糖的释放 每个样品一份等量(提供~1.5 mg)，冷冻干燥，溶解在碳酸氢铵缓冲液(50 mm，pH 8.4)中。将样品放入100oC的加热块中5min变性蛋白质。冷却至室温后，用胰酶和胰凝乳酶处理样品，并在37℃下孵育一夜。每一个胰酶-胰凝乳酶消化液都通过C18Sep pak小柱，用5%的冰醋酸洗脱，然后用20%异丙醇5%冰醋酸、40%异丙醇5%冰醋酸和100%异丙醇串联洗脱。洗脱物最初在氮气气流下干燥，最后冷冻干燥。 干燥的洗脱液用磷酸二氢钠缓冲液溶解，用PNGase F处理，在37oC孵育18h，从多肽链上释放N-连接的多糖。孵育后，酶(PNGase F)消化通过C18Sep pak柱，N-连接的多聚糖组分用5%的冰醋酸洗脱并冷冻干燥。 高效毛细管电泳法分析单糖组成 每个N-连接的葡聚糖组分被分成两个等分：一个用于中性和氨基糖分析，另一个用于唾液酸分析。用于中性糖和氨基糖分析的等价物在100oC下用2N三氟乙酸水解4h，用于唾液酸分析的等价物在80oC用2M醋酸水解3h。然后将水解物冷冻干燥，在水中重新溶解，在冰中超声7分钟，然后转移到注射瓶中。 中性糖和氨基糖的标准混合物，以及已知摩尔数的唾液酸的标准混合物，以与样品相同的方式和同时进行水解。制备了4种浓度的标准混合物(每次注射0.5、1.0、2.0和4.0nmol.)以建立校正方程。样品中每种糖的摩尔数由校正方程中的线性插值法量化。 中性糖、氨基糖和唾液酸的分析采用Dionex DX500系统，系统配有GP40梯度泵、ED40电化学检测器和带有不锈钢针的热分离AS3500自动进样器。中性糖、氨基糖和唾液酸用Dionex CarboPac PA20(3×150 mm)分析柱分离。梯度程序使用洗脱剂A，脱气纳米纯水；B，200 mM NaOH；和C，100 mM NaOH用于中性和氨基糖；以及C和D，1M醋酸钠在100 mM NaOH中用于唾液酸。对于中性糖和氨基糖的测定，每40分钟进行一次注射，对于唾液酸的测定，每35分钟进行一次注射。所有方法都基于Hardy和Townsen(Hardy，M.R.和Townend，R.，“High-pH阴离子交换色层析糖蛋白衍生碳水化合物”，1994，Methods Enzymol)所描述的方案。230：208-225)。仪器控制和数据采集使用Dionex PeakNet软件5.01版完成。

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.