STRUCTRUAL ANALYSIS OF N-LINKED OLIGOSACCHARIDES IN MEN2234 GLYCOPROTEIN

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。 L样品经2N-三氟乙酸水解液在100℃下水解4h，释放出中性糖和氨基糖，水解液冷冻干燥。然后将干燥的样品重新悬浮在150°L水中，并在冰上进行超声波处理。对10例L进行分析。空白(水)被注入，并在标准和样品(数据未显示)之间流动，以防止携带。将标准物质与样品平行水解，然后以三个水平(0.5、1.0和2.0nmol)进样。量化是基于这些标准的线性插值法。 用2M冰醋酸在80℃温和酸解3h，使150份L样品中的唾液酸释放出来。样品如上干燥，再悬浮在150份L水中。对10例L进行分析。空白(水)被注入，并在标准和样品(数据未显示)之间流动，以防止携带。将校准标准与您的样品平行水解，然后以三个水平(0.5、1.0和2.0nmol)注入。量化是基于这些标准的线性插值法。 单糖的分析采用Dionex DX500系统，该系统配备了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。使用Dionex CarboPac PA20(3x150 Mm)分析柱和氨基捕捉柱进行分析。梯度程序使用洗脱剂A，水；B，100 mM氢氧化钠用于中性和氨基糖；C，100 mM氢氧化钠；D，100 mM醋酸钠，100 mM氢氧化钠用于唾液酸。中性糖和氨基糖测定每40分钟注射一次，唾液酸测定每35分钟注射一次。

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.