Grčman Matjaž, Pompe Niko R, Kočar Drago, Pompe Matevž
Faculty of Chemistry and Chemical Technology, University of Ljubljana, 1000 Ljubljana, Slovenia.
Molecules. 2025 Mar 15;30(6):1327. doi: 10.3390/molecules30061327.
The glycosylation process plays a crucial role in the structural integrity and biological activity of glycoproteins, where glycans are attached to a protein backbone. There are many kinds of glycans, the most common being N-glycans, which can be arranged into three classes, that is, complex, hybrid, and high mannoses, forming a structurally very diverse set of polar compounds that are difficult to detect and separate. Most commonly, N-glycans are labeled before separation by charged or fluorescence tags for better MS or fluorescence detection, respectively. This study examines the influence of ionic strength and organic modifier selection on the separation of fluorescently labeled dextran ladders in Hydrophilic Interaction Liquid Chromatography (HILIC). Using a Glycan BEH Amide column and varying the ammonium formate buffer concentration along with acetonitrile and methanol ratios, we investigated analyte retention, separation efficiency, and post-column conductivity changes. Our findings reveal that changes in the ionic strength of the mobile phase do not contribute to changes in selectivity, neither when acetonitrile nor methanol were used as organic modifiers to the mobile phase. However, the addition of methanol significantly changes the separation mechanism where two different prevailing separations mechanisms can be identified. It was assumed that the addition of methanol influences the folding pattern of dextrans around the permanent positive charge on the added tag, which influences the changes of separation selectivity. This work presents a systematic approach to altering mobile phase composition (buffer concentration, organic modifier type) to control retention and selectivity in complex glycan analysis. The discovery that methanol significantly alters separation behavior provides a potential new method for refining HILIC separations of polar compounds.
糖基化过程在糖蛋白的结构完整性和生物活性中起着关键作用，其中聚糖连接到蛋白质主链上。聚糖有多种类型，最常见的是N - 聚糖，它可分为三类，即复杂型、杂合型和高甘露糖型，形成了一组结构非常多样的极性化合物，难以检测和分离。最常见的是，N - 聚糖在分离前用带电或荧光标签进行标记，以便分别进行更好的质谱或荧光检测。本研究考察了离子强度和有机改性剂的选择对亲水作用液相色谱（HILIC）中荧光标记葡聚糖阶梯分离的影响。使用聚糖BEH酰胺柱，改变甲酸铵缓冲液浓度以及乙腈和甲醇的比例，我们研究了分析物保留、分离效率和柱后电导率变化。我们的研究结果表明，当乙腈和甲醇都用作流动相的有机改性剂时，流动相离子强度的变化对选择性变化没有贡献。然而，甲醇的加入显著改变了分离机制，其中可以识别出两种不同的主要分离机制。据推测，甲醇的加入影响了添加标签上永久正电荷周围葡聚糖的折叠模式，从而影响了分离选择性的变化。这项工作提出了一种系统的方法来改变流动相组成（缓冲液浓度、有机改性剂类型），以控制复杂聚糖分析中的保留和选择性。甲醇显著改变分离行为这一发现为改进极性化合物的HILIC分离提供了一种潜在的新方法。
