Université Paris-Saclay, CNRS, Institut Galien Paris Saclay, 92296, Châtenay-Malabry, France; Institute of Pharmaceutical Analysis, College of Pharmacy, Jinan University, Guangzhou, 510632, China.
Translational Glycomics Research Group, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, 10 Egyetem Street, Veszprem, 8200, Hungary.
Anal Chim Acta. 2020 Oct 16;1134:1-9. doi: 10.1016/j.aca.2020.08.002. Epub 2020 Aug 12.
A novel N-glycan enrichment strategy is presented using unexpected but strong interactions between the sulfonate groups brought by the fluorescent dye of glycans and the Zr modified poly(ethylene glycol methacrylate phosphate (EGMP)-co-acrylamide (AM)-co-bis-acrylamide (BAA)) monolith. The poly (EGMP-co-AM-co-BAA) monolith was synthesized via ultraviolet (UV) irradiation and then functionalized with Zr. The obtained monolith was characterized with scanning electron microscopy and mercury intrusion porosimetry. Large through-pores and a continuous skeleton with high permeability were observed. The N-glycans were labeled with the 1-aminopyrene-3, 6, 8-trisulfonic acid (APTS) and enriched by the Zr modified monolith through IMAC interaction. This enrichment step was then coupled off-line to capillary electrophoresis (CE) separation with laser induced fluorescence (LIF) detection. Successful preconcentration of the APTS labeled maltooligosaccharide ladder was achieved under optimized conditions. Enrichment factors obtained for the maltooligosaccharides ranged from 9 to 24 with RSDs from 2.0% to 9.2% (n = 3). Moreover, very good repeatabilities (<6.7%) were obtained for glucose oligomers (4-15 glucose units) corresponding to sizes expected for N-glycans, demonstrating the great potential of this Zr modified monolith to enrich APTS labeled glycans from N-glycoproteins. The proposed method was then successfully applied for the enrichment of N-glycans released from Ribonuclease B, in which case all five expected oligomannose glycans (Man 5 to Man 9) were successfully enriched. Thanks to the advantage of the method to enrich selectively APTS-glycans compared to the commercial SPE columns composed of HILIC or PGC materials, the first proof of concept of on-line enrichment coupled to CE-LIF separation was demonstrated for maltooligosaccharides as well.
提出了一种新颖的 N-糖链富集策略，该策略利用糖的荧光染料带来的磺酸基团与 Zr 修饰的聚（乙二醇甲基丙烯酸磷酸酯（EGMP）-共-丙烯酰胺（AM）-共-双丙烯酰胺（BAA））整体之间的出人意料但很强的相互作用。通过紫外（UV）照射合成了聚（EGMP-co-AM-co-BAA）整体，并对其进行了 Zr 功能化。用扫描电子显微镜和压汞法对获得的整体进行了表征。观察到具有大通孔和高渗透性的连续骨架。用 1-氨基芘-3,6,8-三磺酸（APTS）标记 N-糖，并通过 IMAC 相互作用富集到 Zr 修饰的整体上。然后，将该富集步骤与激光诱导荧光（LIF）检测的毛细管电泳（CE）分离在线耦合。在优化条件下，成功实现了 APTS 标记麦芽寡糖梯的预浓缩。麦芽寡糖的富集因子在 9 到 24 之间，RSD 在 2.0%到 9.2%（n=3）之间。此外，对于葡萄糖低聚物（4-15 个葡萄糖单位），获得了非常好的重复性（<6.7%），这对应于 N-糖的预期大小，证明了这种 Zr 修饰的整体对从核糖核酸酶 B 中释放的 APTS 标记糖的富集具有很大的潜力。然后，该方法成功地应用于从核糖核酸酶 B 中释放的 N-糖的富集，在此情况下，成功地富集了所有五种预期的寡甘露糖糖（Man 5 到 Man 9）。由于该方法相对于由 HILIC 或 PGC 材料组成的商业 SPE 柱具有选择性富集 APTS-糖的优势，因此还首次证明了在线富集与 CE-LIF 分离的概念验证。
