Biomimetic Reagents for the Selective Free Radical and Acid-Base Chemistry of Glycans: Application to Glycan Structure Determination by Mass Spectrometry
Biomimetic Reagents for the Selective Free Radical and Acid-Base Chemistry of Glycans: Application to Glycan Structure Determination by Mass Spectrometry
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DOI：
10.1021/ja402810t
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发表时间：
2013-07-24
影响因子：
15
通讯作者：
Beauchamp, J. L.
中科院分区：
文献类型：
--
作者：
Gao, Jinshan;Thomas, Daniel A.;Beauchamp, J. L.
大自然擅长将聚糖分解成它们的组分，通常是通过酶的酸碱催化来实现糖苷键的选择性切割。注意到质子转移在许多这些酶的活性位点的重要性，我们描述了一种用于酸催化聚糖测序（PRAGS）的隔离质子试剂，该试剂将具有中等质子亲和力的吡啶片段的聚糖还原端衍生化。prags衍生聚糖的气相碰撞活化主要产生C1-O糖苷键裂解，保留还原端上的电荷。由此产生的prags定向的多糖系统解构可以分析提取多糖的组成和序列。多糖还极易被自由基（主要是活性氧）解离，这启发了我们开发一种自由基激活的聚糖测序试剂（FRAGS），该试剂将自由基前体与可偶联到目标聚糖还原末端的吡啶片段结合在一起。frags衍生聚糖的碰撞激活产生自由基，自由基反应产生大量的交叉环裂解、糖苷键裂解以及这些类型的裂解的组合，并在还原端保留电荷。用FRAGS试剂通过仅在这些位置观察到的特定碎片模式来识别分支位点。研究和讨论了解离机理以及试剂在线性和高支链聚糖结构分析中的应用。与早期采用质谱法的研究相比，本文开发的聚糖结构分析方法具有独特的优势。
Nature excels at breaking down glycans into their components, typically via enzymatic acid-base catalysis to achieve selective cleavage of the glycosidic bond. Noting the importance of proton transfer in the active site of many of these enzymes, we describe a sequestered proton reagent for acid-catalyzed glycan sequencing (PRAGS) that derivatizes the reducing terminus of glycans with a pyridine moiety possessing moderate proton affinity. Gas-phase collisional activation of PRAGS-derivatized glycans predominately generates C1-O glycosidic bond cleavages retaining the charge on the reducing terminus. The resulting systematic PRAGS-directed deconstruction of the glycan can be analyzed to extract glycan composition and sequence. Glycans are also highly susceptible to dissociation by free radicals, mainly reactive oxygen species, which inspired our development of a free radical activated glycan sequencing (FRAGS) reagent, which combines a free radical precursor with a pyridine moiety that can be coupled to the reducing terminus of target glycans. Collisional activation of FRAGS-derivatized glycans generates a free radical that reacts to yield abundant cross-ring cleavages, glycosidic bond cleavages, and combinations of these types of cleavages with retention of charge at the reducing terminus. Branched sites are identified with the FRAGS reagent by the specific fragmentation patterns that are observed only at these locations. Mechanisms of dissociation as well as application of the reagents for both linear and highly branched glycan structure analysis are investigated and discussed. The approach developed here for glycan structure analysis offers unique advantages compared to earlier studies employing mass spectrometry for this purpose.