Top-down approach for the direct characterization of low molecular weight heparins using LC-FT-MS.
Top-down approach for the direct characterization of low molecular weight heparins using LC-FT-MS.
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DOI：
10.1021/ac302232c
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发表时间：
2012-10-16
影响因子：
7.4
通讯作者：
Linhardt, Robert J.
中科院分区：
文献类型：
--
作者：
Li, Lingyun;Zhang, Fuming;Zaia, Joseph;Linhardt, Robert J.
低分子肝素(LMWHs)是一种结构复杂、多相、多分散、高负电荷的多糖类混合物。低分子量氢化合物的直接表征是目前可用的分析技术的主要挑战。电喷雾电离(ESI)液-质联用(LC-MS)是蛋白质组、代谢组学和糖组学领域中表征复杂生物样品的有力工具。将LC-MS用于肝素低聚糖的分析，采用尺寸排斥、反相离子对层析和芯片基酰胺亲水作用层析(HILIC)对其进行分离。然而，ESI-LC-MS由于其结构复杂、电离效率低和硫酸盐损失等原因，在直接表征完整的低分子量氢化合物(自顶向下分析)方面的应用一直受到限制。在这里，我们提出了一个简单而可靠的HILIC-傅立叶变换(FT)-ESI-MS平台，使用自上而下的方法来表征和比较目前市场上的两种LMWH产品，不需要特殊的样品制备步骤。这种HILIC系统依赖于交联二醇而不是酰胺化学，在流动相中使用相对较高比例的乙腈提供高分辨率的色谱分离，从而实现稳定和高效的电离。使用生物信息学软件(GlycerSoft 1.0)自动分配质量精度在5ppm以内的结构。
Low molecular heparins (LMWHs) are structurally complex, heterogeneous, polydisperse, and highly negatively charged mixtures of polysaccharides. The direct characterization of LMWH is a major challenge for currently available analytical technologies. Electrospray ionization (ESI) liquid chromatography-mass spectrometry (LC-MS) is a powerful tool for the characterization complex biological samples in the fields of proteomics, metabolomics and glycomics. LC-MS has been applied to the analysis of heparin oligosaccharides, separated by size exclusion, reversed phase ion-pairing chromatography and by chip-based amide hydrophilic interaction chromatography (HILIC). However, there have been limited applications of ESI-LC-MS for the direct characterization of intact LMWHs (top-down analysis) due to their structural complexity, low ionization efficiency and sulfate loss. Here we present a simple and reliable HILIC-Fourier transform (FT)-ESI-MS platform to characterize and compare two currently marketed LMWH products using the top-down approach requiring no special sample preparation steps. This HILIC system relies on cross-linked diol rather than amide chemistry, affording highly resolved chromatographic separations using a relatively high percentage of acetonitrile in the mobile phase, resulting in stable and high efficiency ionization. Bioinformatics software (GlycerSoft 1.0) was used to automatically assign structures within 5-ppm mass accuracy.
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