Size-exclusion chromatography of heparin oligosaccharides at high and low pressure
Size-exclusion chromatography of heparin oligosaccharides at high and low pressure
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DOI：
10.1016/j.jchromb.2006.04.013
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发表时间：
2006-06-06
影响因子：
3
通讯作者：
Zaia, J
中科院分区：
文献类型：
--
作者：
Ziegler, A;Zaia, J
最近关于糖胺聚糖（GAGs）的特异性和非特异性相互作用的发现强调了它们在生物学中的关键作用，并呼吁改进测序工具。本研究在高压和低压下评估肝素寡糖的尺寸排除色谱（SEC），所需的量低至0.2微克，使用常规紫外检测，在肝素解聚酶解聚后。由于肝素寡糖在生理pH下的高电荷，其SEC洗脱量取决于分子大小和基质的电荷排斥。因此，GAGs的SEC洗脱体积小于类似分子量的球状蛋白，这一点可能被利用。因此，较大的肝素寡糖在高离子强度流动相（bbb30 mM）下，根据其大小，分离效果最好；相比之下，双糖在低离子强度下根据其电荷进行最佳分离，与在线耦合质谱相兼容。优化的SEC提供了在还原端含有醛酸的特征肝素三糖的分离，并提示肝素作为游离聚糖的细胞储存。(c) 2006 Elsevier B.V.版权所有
Recent findings on specific and non-specific interactions of glycosaminoglycans (GAGs) accentuate their pivotal role in biology and the call for improved sequencing tools. The present study evaluates size-exclusion chromatography (SEC) of heparin oligosaccharides at high and low pressure, requiring amounts as low as 0.2 microgram, using conventional UV detection after depolymerization with heparin lyases. Because of their high charge at physiological pH, SEC elution volumes of heparin oligosaccharides depend on both molecular size and charge repulsion from the matrix. As a consequence, SEC elution volumes of GAGs are smaller than those of globular proteins of similar molecular weight, and this might be exploited. Accordingly, larger heparin oligosaccharides are best separated according to their size at high ionic strength of the mobile phase (> 30 mM); in contrast, disaccharides are best separated according to their charge at low ionic strength, compatible with on-line coupling to mass spectrometry. Optimized SEC affords separation of characteristic heparin trisaccharides that contain uronic acid at the reducing end and suggest cellular storage of heparin as a free glycan. (c) 2006 Elsevier B.V. All rights reserved.