STRUCTRUAL ANALYSIS OF N-LINKED OLIGOSACCHARIDES IN MEN2234 GLYCOPROTEIN

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。 通过在100 ℃下用2 N三氟乙酸水解4 h，从150 µ L每份提交的样品中释放中性糖和氨基糖。 水解后，将样品冻干。 然后将干燥的样品重悬于150 L水中，并在冰上超声处理。 为了分析，进样10 μ L。 注入空白（水），并在标准品和样品之间运行（数据未显示），以防止残留。 校准标准品与样品平行水解，然后以3个水平（0.5、1.0和2.0 nmol）进样。定量基于这些标准品的线性插值。 通过使用2 M乙酸在80 ℃下温和酸水解3 h，从150 µ L的每份样品中释放唾液酸。 如上所述干燥样品，并重悬于150 L水中。 为了分析，进样10 μ L。 注入空白（水），并在标准品和样品之间运行（数据未显示），以防止残留。 校准标准品与样品平行水解，然后以三个水平（0.5、1.0和2.0 nmol）进样。 定量基于这些标准品的线性插值。 使用配备有GP 40梯度泵、ED 40电化学检测器和含有不锈钢针的Thermo-Separations AS 3500自动进样器的Dionex DX 500系统分析单糖。 使用Dionex CarboPac PA 20（3 x 150 mm）分析色谱柱和Amino Trap色谱柱进行分析。 梯度程序使用洗脱液A，水; B，100 mM NaOH用于中性和氨基糖; C，100 mM NaOH;和D，100 mM NaOH中的1 M乙酸钠用于唾液酸。中性糖和氨基糖测定每40分钟进样一次，唾液酸测定每35分钟进样一次。

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. Neutral and amino sugars were released from 150 ¿L of each of the samples submitted through hydrolysis with 2 N trifluoroacetic acid at 100¿C for 4 h. After hydrolysis the samples were lyophilized. The dried samples were then resuspended in 150 ¿L water and sonicated on ice. For analysis, 10 ¿L were injected. Blanks (water) were injected and run between the standards and the sample (data not shown) to prevent carry-over. The calibration standards were hydrolyzed in parallel with the samples, and then injected at three levels (0.5, 1.0 and 2.0 nmol). The quantitation was based on a linear interpolation from these standards. Sialic acids were released from 150 ¿L of each of the samples submitted by mild acid hydrolysis using 2M acetic acid at 80¿C for 3 h. The samples were dried as above, and resuspended in 150 ¿L water. For analysis, 10 ¿L were injected. Blanks (water) were injected and run between the standards and the sample (data not shown) to prevent carry-over. The calibration standards were hydrolyzed in parallel with your samples, and then injected at three levels (0.5, 1.0 and 2.0 nmol). The quantitation was based on a linear interpolation from these standards. Monosaccharides were analyzed using a Dionex DX500 system equipped with a GP40 gradient pump, an ED40 electrochemical detector and a Thermo-Separations AS3500 autosampler containing a stainless steel needle. Analyses were performed using a Dionex CarboPac PA20 (3 x 150 mm) analytical column with an Amino Trap column. The gradient programs used eluents A, water; B, 100 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.