Institut Galien Paris Sud, UMR 8612, Protein and Nanotechnology in Analytical Science (PNAS), CNRS, Univ. Paris-Sud, Univ. Paris-Saclay, 5 rue Jean Baptiste Clément, 92290 Châtenay-Malabry, France.
Université Paris-Saclay, INSERM UMR1193, Mécanismes cellulaires et moléculaires de l'adaptation au stress et cancérogenèse, Châtenay-Malabry, France; AP-HP, Biochimie Métabolique et Cellulaire, Hôpital Bichat-Claude Bernard, Paris, France.
J Chromatogr A. 2021 Nov 8;1657:462593. doi: 10.1016/j.chroma.2021.462593. Epub 2021 Oct 2.
We present in this study a novel strategy to drastically improve the detection sensitivity and peak capacity for capillary electrophoresis with laser induced fluorescent detection (CE-LIF) of glucose oligomers and released glycans. This is based on a new approach exploiting a polymer-free background electrolyte (BGE) for CE-LIF of glycans. The best performance in terms of sample stacking and suppression of electroosmotic flow (EOF) was found for a BGE composed of triethanolamine/citric acid and triethanolamine/acetic acid at elevated ionic strengths (IS up to 200 mM). Compared to the conventional protocols for CE-LIF of glucose-oligosaccharides and released glycans, our polymer-free strategy offered up to 5-fold improvement of detection sensitivity and visualization of higher degree of polymerization (DP) of glucose oligomers (18 vs 15). To further improve the detection sensitivity, a new electrokinetic preconcentration strategy via large volume sample stacking with electroosmotic modulation without having recourse to neutrally coated capillaries is proposed, offering a 200-fold signal enhancement. This approach is based on variation of the buffer's IS, rather than pH adjustment as in conventional methods, for EOF modulation or quasi-total reduction. This strategy allows selecting with high flexibility the best pH conditions to perform efficient preconcentration and separation. The new approach was demonstrated to be applicable for the analysis of N-linked oligosaccharides released from a model glycoprotein (Human Immunoglobulin G) and applied to map N-glycans from human serum for congenital disorders of glycosylation (CDG) diagnosis.
我们在这项研究中提出了一种新颖的策略，可极大地提高毛细管电泳与激光诱导荧光检测（CE-LIF）检测葡萄糖低聚物和释放糖的检测灵敏度和峰容量。这是基于一种新方法，该方法利用无聚合物背景电解质（BGE）来进行 CE-LIF 糖分析。在提高的离子强度（高达 200 mM）下，发现三乙醇胺/柠檬酸和三乙醇胺/乙酸组成的 BGE 在样品堆积和抑制电渗流（EOF）方面具有最佳性能。与传统的 CE-LIF 葡萄糖低聚糖和释放糖的方案相比，我们的无聚合物策略将检测灵敏度提高了 5 倍，并可视化了更高聚合度（DP）的葡萄糖低聚物（18 与 15）。为了进一步提高检测灵敏度，提出了一种新的电动浓缩策略，通过大体积样品堆积和电渗流调制而无需使用中性涂层毛细管，从而实现了 200 倍的信号增强。该方法基于缓冲液离子强度的变化，而不是像常规方法那样通过 pH 调节来调节 EOF 或准完全减少。这种策略可以灵活地选择最佳的 pH 条件，以实现有效的浓缩和分离。该新方法适用于从模型糖蛋白（人免疫球蛋白 G）释放的 N-连接寡糖的分析，并应用于分析人血清中的 N-糖链以诊断先天性糖基化障碍（CDG）。
