A versatile strategy for high-resolution separation of reducing glycan mixtures as hydrazones by two-dimensional high-performance liquid chromatography
A versatile strategy for high-resolution separation of reducing glycan mixtures as hydrazones by two-dimensional high-performance liquid chromatography
复制标题
通过二维高效液相色谱高分辨率分离腙等还原性聚糖混合物的通用策略
DOI：
10.1016/j.chroma.2022.463599
复制
发表时间：
2022-11-04
影响因子：
4.1
通讯作者：
Wang，Zhongfu
中科院分区：
文献类型：
--
作者：
Wang，Chengjian;Gao，Xi;Wang，Zhongfu
糖组学分析由于缺乏结构确定的单个聚糖作为模型化合物而受到破坏。然而，从天然资源中制备单个聚糖具有挑战性，主要是由于天然存在的聚糖的高度多样的结构、复杂的混合物形式和无发色团的性质引起的分离困难。在这项研究中，我们报告了一个简单的，通用的和低成本的聚糖分离策略，glycoselection，它允许通过可逆的显色衍生化的酰肼化学结合二维高效液相色谱法（2D-HPLC）从它们的混合物中制备单个还原聚糖。以乳糖和麦芽糖糊精为模型聚糖，考察了反应条件，结果表明：可逆酰肼标记和一锅法腙转化反应在温和条件下是可行的;酰肼衍生物在溶液中稳定性好; 7种肼类显色剂在聚糖标记和柱后脱标签过程中的产率存在差异。建立了麦芽糊精的二维高效液相色谱分离条件，最终获得了十四种高纯度的还原性低聚葡聚糖。使用这种策略，我们还成功地制备和鉴定了11个来自鸡卵清蛋白的中性还原性N-聚糖和13个来自人乳的中性还原性寡糖和8个单独的唾液酸化寡糖，证明了其对不同类型的还原性聚糖以及生物样品的良好适用性。鉴于单个还原性聚糖与几乎所有聚糖衍生化方案和聚糖分析技术的兼容性以及该方法的大规模应用潜力，本工作为天然聚糖的化合物特异性分析提供了通用方法，对糖组学研究具有重要意义。
Glycomics analysis has been undermined by the lack of structurally defined individual glycans as model compounds. However, it is challenging to prepare individual glycans from natural resources, mainly due to separation difficulties caused by highly diverse structure, complicated mixture form and chromophore-free property of naturally-existing glycans. In this study, we report a simple, universal and low-cost glycan separation strategy, glycoselection, which allows preparation of individual reducing glycans from their mixtures through reversible chromogenic derivatization by hydrazide chemistry in combination with two-dimensional high-performance liquid chromatography (2D-HPLC). Investigations on reaction conditions using lactose and maltodextrin as model glycans showed the feasibility of reversible hydrazide labeling and one-pot hydrazone conversion under mild conditions, the good stability of hydrazone-form derivatives of glycans in solution and the difference among seven selected hydrazine-carrying chromogenic reagents in product yields during glycan labeling and post-column detagging. The 2D-HPLC separation conditions were established on maltodextrin, from which fourteen highly-purified individual reducing oligo-glucans were ultimately obtained. Using this strategy, we also successfully prepared and identified eleven individual neutral reducingN-glycans from chicken ovalbumin and thirteen individual neutral reducing oligosaccharides and eight individual sialylated ones from human milk, demonstrating its good applicability to different types of reducing glycans as well as biological samples. Given the compatibility of individual reducing glycans with almost all of glycan derivatization protocols and analytical techniques of glycans and the potential of the method for larger scale application, this work provides a universal approach to compound-specific analysis of natural glycans and has great significance for glycomics studies.