An ultrasensitive chemical method for polysialic acid analysis
An ultrasensitive chemical method for polysialic acid analysis
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DOI：
10.1093/glycob/11.9.759
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发表时间：
2001-09-01
期刊：
影响因子：
4.3
通讯作者：
Inoue, Y
中科院分区：
文献类型：
--
作者：
Inoue, S;Lin, SL;Inoue, Y
建立了一种利用荧光辅助高效液相色谱法分析聚唾液酸（polySia）链的超灵敏方法。新的方法是一个实质性的改进，我们早期的方法，其中还原性末端Sia残基的同源系列的寡/polySia水解过程中衍生化反应释放的同时标记的荧光试剂，1，2-二氨基-4，5-亚甲二氧基苯（DMB）原位。我们首先广泛地研究了oligo/polySia在用于1，2-二氨基-4，5-亚甲二氧基苯衍生化的酸（0.02 M三氟乙酸）中在各种反应时间和温度条件下的稳定性，通过高效阴离子交换色谱-脉冲电化学检测（HPAEC-PED）分析水解产物。然后，我们优化了反应条件，以最大限度地减少母体polySia的降解，同时保持高的衍生化速率。使用DNAPac PA-100色谱柱而不是MonoQ色谱柱，实现了高达DP 90的polySia峰的基线分离度，每个分离峰的检测阈值为1.4飞摩尔。用该方法分析了鸡胚脑中含有聚Sia的糖肽组分的聚合度，并与HPAEC-PED的结果进行了比较。
An ultrasensitive method for analysis of polysialic acid (polySia) chains, using fluorescence-assisted high-performance liquid chromotography was developed. The new method is a substantial improvement of our earlier method in which the reducing terminal Sia residues of a homologous series of oligo/polySia hydrolytically released during derivatization reaction were simultaneously labeled with a fluorogenic reagent, 1,2-diamino-4,5-methylenedioxybenzene (DMB) in situ. We first studied extensively the stability of oligo/polySia in the acid (0.02 M trifluoracetic acid) used for 1,2-diamino-4,5-methylenedioxybenzene derivatization under various conditions of reaction time and temperature, analyzing the hydrolytic products by high-performance anion exchange chromatography with pulsed electrochemical detection (HPAEC-PED). Then we optimized the reaction conditions to minimize degradation of the parent polySia while maintaining high derivatization rate. Using a DNAPac PA-100 column rather than a MonoQ column, baseline resolution of polySia peaks up to DP 90 with a detection threshold of 1.4 femtomol per resolved peak was achieved. The new method was used to analyze the degree of polymerization of a polySia-containing glycopeptide fraction derived from embryonic chicken brain, and the results were compared with those obtained by HPAEC-PED.