In-Depth Characterization of N-Linked Oligosaccharides Using Fluoride-Mediated Negative Ion Microfluidic Chip LC-MS
In-Depth Characterization of N-Linked Oligosaccharides Using Fluoride-Mediated Negative Ion Microfluidic Chip LC-MS
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DOI：
10.1021/ac3031898
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发表时间：
2013-03-19
影响因子：
7.4
通讯作者：
Karger, Barry L.
中科院分区：
文献类型：
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作者：
Ni, Wenqin;Bones, Jonathan;Karger, Barry L.
利用填充有多孔石墨化碳(PGC)的微流控芯片对N-葡聚糖进行高效液相色谱-电喷雾电离(ESI)串联质谱仪(LC-MS/MS)表征，是寡糖分析中一个快速发展的领域。阳离子ESI-MS在碰撞诱导解离(CID)作用下产生B/Y型糖苷碎片离子。虽然这些离子有助于多糖测序，但它们提供的关于连接和位置异构体的信息很少。在这些情况下，异构体的鉴定是通过保留在PGC固定相上，在那里，原则上可以分离特定的结构异构体。在本文中，我们通过实现氟介导的负ESI-MS来扩大PGC微流控芯片/MS平台的适用性。添加到流动相中的氟化铵有助于形成假分子低聚糖阴离子，这是因为氟化物能够从多糖结构中提取质子。负电荷导致生成C型糖苷片段、信息量高的A型跨环片段离子和额外的气相离子反应产物(例如，D-和E-型离子)，当这些离子结合在一起时，导致低聚糖的深入表征，包括连接和位置异构体。由于通过PGC相分离异构体，将具有完整还原末端的低聚糖与其实验制备的相应的糖醇进行了比较，发现具有自由还原末端的寡糖进行了更灵敏的MS，特别是MS/MS分析。氟化物还确保了带电低聚糖从PGC固定相中的回收。应用于多克隆人和小鼠血清免疫球蛋白释放的N-糖链的表征，证明了芯片/阴性ESI方法的有效性。
Characterization of N-glycans by liquid chromatography-positive electrospray ionization (ESI) tandem mass spectrometry (LC-MS/MS) using a microfluidic chip packed with porous graphitized carbon (PGC) represents a rapidly developing area in oligosaccharide analysis. Positive ion ESI-MS generates B/Y-type glycosidic fragment ions under collisional-induced dissociation (CID). Although these ions facilitate glycan sequencing, they provide little information on linkage and positional isomers. Isomer identification in these cases is by retention on the PGC stationary phase where the specific structural isomers can, in principle, be separated. In this paper, we broaden the applicability of the PGC microfluidic chip/MS platform by implementing fluoride-mediated negative ESI-MS. Ammonium fluoride, added to the mobile phase, aids in the formation of pseudomolecular oligosaccharide anions due to the ability of fluoride to abstract a proton from the glycan structure. The negative charge results in the generation of C-type glycosidic fragments, highly informative A-type cross-ring fragment ions, and additional gas-phase ion reaction products (e.g., D- and E-type ions), which, when combined, lead to in-depth oligosaccharide characterization, including linkage and positional isomers. Due to the separation of anomers by the PGC phase, comparison of oligosaccharides with an intact reducing terminus to their experimentally prepared corresponding alditols was performed, revealing a more sensitive MS and, especially, MS/MS analysis from the glycans with a free reducing end. Fluoride also ensured recovery of charged oligosaccharides from the PGC stationary phase. Application to the characterization of N-glycans released from polyclonal human and murine serum IgG is presented to demonstrate the effectiveness of the chip/negative ESI approach.