Versatile Separation and Analysis of Heparan Sulfate Oligosaccharides Using Graphitized Carbon Liquid Chromatography and Electrospray Mass Spectrometry
Versatile Separation and Analysis of Heparan Sulfate Oligosaccharides Using Graphitized Carbon Liquid Chromatography and Electrospray Mass Spectrometry
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DOI：
10.1021/acs.analchem.7b01417
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发表时间：
2017-09-05
影响因子：
7.4
通讯作者：
Karlsson, Niclas
中科院分区：
文献类型：
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作者：
Miller, Rebecca L.;Guimond, Scott E.;Karlsson, Niclas
肝素和硫酸乙酰肝素（HS）本质上含有多种异构体结构，这是调节生物过程的基础。在这里，我们报告使用的多孔石墨化碳（PGC）的LC-MS方法与有效的分离和灵敏度，以分离消化HS低聚糖的混合物。该方法的应用允许分离具有不同聚合度（DP）的低聚糖混合物，聚合度范围从DP 4到DP 8，两种基线分辨的DP 4异构体，四种DP 6异构体，并观察到DP 3低聚糖。复杂混合物的PGC LC-MS表明，化合物以亲水性的降序从柱中洗脱，首先洗脱的是更高度硫酸化的结构。我们的数据表明硫酸化水平、链长和构象都影响洗脱顺序。我们发现PGC对dp 4和dp 6异构体结构的分辨能力使得这种方法对于HS的测序特别有用，因为缺乏污染的异构体结构提供了来自MS/MS数据的明确的结构归属。总的来说，这项工作表明，PGC柱为基础的方法是强大的工具，用于增强分离和分析的异质混合物的HS糖物种。
Heparin and heparan sulfate (HS) by nature contain multiple isomeric structures, which are fundamental for the regulation of biological processes. Here we report the use of a porous graphitized carbon (PGC) LC-MS method with effective separation and sensitivity to separate mixtures of digested HS oligosaccharides. Application of this method allowed the separation of oligosaccharide mixtures with various degree of polymerization (dp) ranging from dp4 to dp8, two dp4 isomers that were baseline resolved, four dp6 isomers, and the observation of a dp3 oligosaccharide. PGC LC-MS of complex mixtures demonstrated that compounds eluted from the column in decreasing order of hydrophilicity, with the more highly sulfated structures eluting first. Our data indicate that sulfation levels, chain length, and conformation all effect elution order. We found that PGC's resolving capabilities for the dp4 and dp6 isomeric structures makes this methodology particularly useful for the sequencing of HS saccharides, because the lack of contaminating isomeric structures provides unambiguous structural assignments from the MS/MS data. Collectively this work demonstrates that PGC column-based methods are powerful tools for enhanced separation and analysis of heterogeneous mixtures of HS saccharide species.