The structures of glycosaminoglycans (GAGs), especially the patterns of modification, are crucial to modulate interactions with various protein targets. It is very challenging to determine the fine structures using liquid chromatography-mass spectrometry (LC-MS) due in large part to the gas-phase sulfate losses upon collisional activation. Previously, our group reported a method for fine structure analysis that required permethylation of the GAG oligosaccharide. However, uncontrolled depolymerization during the permethylation process due to esterification of uronic acid lowers the reliability of the method to resolve structures of GAGs, especially for larger oligosaccharides. Here, we describe a simplified derivatization method using propionylation and desulfation. The oligosaccharides have all hydroxyl and amine groups protected with propionyl groups and then have sulfate groups removed to generate unprotected hydroxyl and amine groups at all sites that were previously sulfated. This derivatized oligosaccharide generates informative fragments during collision-induced dissociation that resolve the original sulfation patterns. This method is demonstrated to enable accurate determination of sulfation patterns of even the highly sulfated pentasaccharide fondaparinux by MS2 and MS3. Using a mixture of dp6 from porcine heparin, we demonstrate that this method allows for structural characterization of complex mixtures, including clear chromatographic separation and sequencing of structural isomers, all at high yields without evidence of depolymerization. This represents a marked improvement in the reliability to structurally characterize GAG oligosaccharides over permethylation-based derivatization schemes.

中文翻译：

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过酰化与串联质谱联用，在不解聚的情况下对硫酸化糖胺聚糖混合物进行结构测序。

糖胺聚糖 (GAG) 的结构，尤其是修饰模式，对于调节与各种蛋白质靶标的相互作用至关重要。使用液相色谱-质谱法 (LC-MS) 确定精细结构非常具有挑战性，这在很大程度上是由于碰撞活化时气相硫酸盐的损失。此前，我们小组报道了一种精细结构分析方法，该方法需要 GAG 寡糖的全甲基化。然而，由于糖醛酸的酯化，全甲基化过程中不受控制的解聚降低了解析 GAG 结构的方法的可靠性，尤其是对于较大的寡糖。在这里，我们描述了一种使用丙酰化和脱硫的简化衍生方法。低聚糖的所有羟基和胺基都用丙酰基保护，然后去除硫酸根，在所有先前被硫酸化的位点上生成未保护的羟基和胺基。这种衍生化的寡糖在碰撞诱导解离过程中产生信息片段，解决原始硫酸化模式。该方法经证明能够通过 MS2 和 MS3 准确测定甚至高度硫酸化的五糖磺达肝素的硫酸化模式。使用来自猪肝素的 dp6 混合物，我们证明该方法允许复杂混合物的结构表征，包括清晰的色谱分离和结构异构体的测序，所有这些都以高产率进行，没有解聚的证据。