Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, VA 23219, USA.
Electrophoresis. 2011 Nov;32(21):3070-7. doi: 10.1002/elps.201100175. Epub 2011 Oct 17.
Glycosaminoglycan (GAG) analysis represents a challenging frontier despite the advent of many high-resolution technologies because of their unparalleled structural complexity. We previously developed a resolving agent-aided capillary electrophoretic approach for fingerprinting low-molecular-weight heparins (LMWHs) to profile their microscopic differences and assess batch-to-batch variability. In this report, we study the application of this approach for fingerprinting other GAGs and analyze the basis for the fingerprints observed in CE. Although the resolving agents, linear polyalkylamines, could resolve the broad featureless electropherogram of LMWH into a large number of distinct, highly reproducible peaks, longer GAGs such as chondroitin sulfate, dermatan sulfate, and heparin responded in a highly individualistic manner. Full-length heparin interacted with linear polyalkylamines very strongly followed by dermatan sulfate, whereas chondroitin sulfate remained essentially unaffected. Oversulfated chondroitin sulfate could be easily identified from full-length heparin. Scatchard analysis of the binding profile of enoxaparin with three linear polyalkylamines displayed a biphasic binding profile suggesting two distinctly different types of interactions. Some LMWH chains were found to interact with linear polyalkylamines with affinities as high as 10 nM, whereas others displayed nearly 5000-fold weaker affinities. These observations provide fundamental insight into the basis for fingerprinting of LMWHs by linear polyalkylamine-based resolving agents, which could be utilized in the design of advanced resolving agents for compositional profiling, direct sequencing, and chemoinformatics studies.
糖胺聚糖 (GAG) 分析是一个极具挑战性的领域，尽管出现了许多高分辨率技术，但由于其无与伦比的结构复杂性，仍然具有挑战性。我们之前开发了一种基于解析剂的毛细管电泳方法，用于对低分子量肝素 (LMWH) 进行指纹图谱分析，以分析其微观差异并评估批间变异性。在本报告中，我们研究了该方法在对其他 GAG 进行指纹图谱分析中的应用，并分析了在 CE 中观察到的指纹图谱的基础。虽然解析剂，即线性聚烷基胺，可以将 LMWH 的宽无特征的电泳图谱解析为大量独特且高度可重复的峰，但更长的 GAG 如硫酸软骨素、硫酸皮肤素和肝素的反应则非常个性化。全长肝素与线性聚烷基胺强烈相互作用，其次是硫酸皮肤素，而硫酸软骨素基本不受影响。过度硫酸化的硫酸软骨素可以很容易地从全长肝素中识别出来。依诺肝素与三种线性聚烷基胺的结合曲线的 Scatchard 分析显示出双相结合曲线，表明存在两种截然不同的相互作用类型。发现一些 LMWH 链与线性聚烷基胺的相互作用亲和力高达 10 nM，而其他链的亲和力则弱了近 5000 倍。这些观察结果为基于线性聚烷基胺的解析剂对 LMWH 进行指纹图谱分析的基础提供了基本的见解，这可以用于设计用于组成分析、直接测序和化学信息学研究的先进解析剂。
