Peracylation Coupled with Tandem Mass Spectrometry for Structural Sequencing of Sulfated Glycosaminoglycan Mixtures without Depolymerization.
Peracylation Coupled with Tandem Mass Spectrometry for Structural Sequencing of Sulfated Glycosaminoglycan Mixtures without Depolymerization.
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DOI：
10.1021/jasms.0c00178
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发表时间：
2020-10-07
影响因子：
3.2
通讯作者：
Sharp JS
中科院分区：
文献类型：
--
作者：
Liu H;Liang Q;Sharp JS
糖胺多聚糖(GAG)的结构，特别是修饰模式，对调节与各种蛋白质靶标的相互作用至关重要。很大程度上由于碰撞活化时气相硫酸盐的损失，用液相色谱-质谱仪(LC-MS)测定精细结构是非常困难的。此前，我们小组报告了一种精细结构分析的方法，该方法需要GAG寡糖的全甲基化。然而，糖醛酸在全甲基化过程中由于酯化而不受控制地解聚，降低了该方法解析GAG结构的可靠性，特别是对于较大的寡糖。在这里，我们描述了一种简化的使用丙酰化和脱硫的衍生化方法。低聚糖具有丙酰基保护的所有羟基和胺基，然后将硫酸盐基团移除，以在先前硫酸盐化的所有位置生成不受保护的羟基和胺基。这种衍生的低聚糖在CID过程中产生信息片段，解析原始的硫酸盐化模式。这种方法被证明能够用MS2和MS3准确地确定即使是高度硫酸化的五糖磺胺肝素的硫化模式。使用猪肝素的DP6混合物，我们证明了这种方法允许对复杂混合物的结构进行表征，包括清晰的色层分离和结构异构体的测序，所有这些都是在没有解聚证据的情况下高产率地进行的。这表明，与基于全甲基化的衍生化方案相比，在结构表征GAG寡糖方面的可靠性有了显着的提高。
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, 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 CID 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 yield without evidence of depolymerization. This represents a marked improvement in reliability to structurally characterize GAG oligosaccharides over permethylation-based derivatization schemes.
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影响因子：
7
作者：
Chiu, Yulun;Huang, Rongrong;Sharp, Joshua S.
通讯作者：
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影响因子：
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作者：
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DOI：
10.1007/s13361-011-0174-0
发表时间：
2011-09
影响因子：
3.2
作者：
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通讯作者：
Sharp, Joshua S.
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影响因子：
4.6
作者：
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通讯作者：
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