Rohrer J S, Thayer J, Weitzhandler M, Avdalovic N
Dionex Corporation, 445 Lakeside Drive, Sunnyvale, CA 94088, USA.
Glycobiology. 1998 Jan;8(1):35-43. doi: 10.1093/glycob/8.1.35.
Presence or absence of N-acetylneuraminic acid (Neu5Ac) can change a sialylated glycoprotein's serum half-life and possibly its function. We evaluated the linearity, sensitivity, reproducibility, and accuracy of a HPAEC/PAD method to determine its suitability for routine simultaneous analysis of Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). An effective internal standard for this analysis is 3-deoxy-d-glycero-d-galacto-2-nonulosonic acid (KDN). We investigated the effect of the Au working electrode recession and determined that linear range and sensitivity were dependent on electrode recession. Using an electrode that was 350 microm recessed from the electrode block, the minimum detection limits of Neu5Ac, KDN, and Neu5Gc were 2, 5, and 2 pmol, respectively, and were reduced to 1, 2, and 0.5 pmol using a new electrode. The response of standards was linear from 10 to 500 pmol (r2>0.99) regardless of electrode recession. When Neu5Ac, KDN, and Neu5Gc (200 pmol each) were analyzed repetitively for 48 h, area RSDs were <3%. Reproducibility was unaffected when injections of glycoprotein neuraminidase and acid digestions were interspersed with standard injections. Area RSDs of Neu5Ac and Neu5Gc improved when the internal standard was used. We determined the precision and accuracy of this method for both a recessed and a new working electrode by analyzing Neu5Ac and Neu5Gc contents of bovine fetuin and bovine and human transferrins. Results were consistent with published values and independent of the working electrode. The sensitivity, reproducibility, and accuracy of this method make it suitable for direct routine analysis of glycoprotein Neu5Ac and Neu5Gc contents.
N-乙酰神经氨酸（Neu5Ac）的存在与否会改变唾液酸化糖蛋白的血清半衰期，并可能影响其功能。我们评估了一种高效阴离子交换色谱-脉冲安培检测（HPAEC/PAD）方法的线性、灵敏度、重现性和准确性，以确定其是否适合同时对Neu5Ac和N-羟乙酰神经氨酸（Neu5Gc）进行常规分析。该分析的有效内标物是3-脱氧-D-甘油-D-半乳糖-2-壬酮糖酸（KDN）。我们研究了金工作电极凹陷的影响，确定线性范围和灵敏度取决于电极凹陷。使用从电极块凹陷350微米的电极时，Neu5Ac、KDN和Neu5Gc的最低检测限分别为2、5和2皮摩尔，而使用新电极时可降至1、2和0.5皮摩尔。无论电极凹陷情况如何，标准品的响应在10至500皮摩尔范围内呈线性（r2>0.99）。当对Neu5Ac、KDN和Neu5Gc（各200皮摩尔）进行48小时的重复分析时，面积相对标准偏差（RSD）<3%。当糖蛋白神经氨酸酶注射和酸消化与标准品注射穿插进行时，重现性不受影响。使用内标物时，Neu5Ac和Neu5Gc的面积RSD有所改善。我们通过分析牛胎球蛋白、牛和人转铁蛋白中的Neu5Ac和Neu5Gc含量，确定了该方法对于凹陷工作电极和新工作电极的精密度和准确性。结果与已发表的值一致，且与工作电极无关。该方法的灵敏度、重现性和准确性使其适用于直接常规分析糖蛋白中Neu5Ac和Neu5Gc的含量。
