Auer Felicia, Guttman Andras
Translational Glycomics Research Group, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Veszprem, Hungary.
Translational Glycomics Research Group, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Veszprem, Hungary; Horváth Csaba Memorial Laboratory of Bioseparation Sciences, Research Center for Molecular Medicine, Faculty of Medicine, University of Debrecen, Hungary.
Talanta. 2025 May 15;287:127601. doi: 10.1016/j.talanta.2025.127601. Epub 2025 Jan 15.
Sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) is a frequently used analytical technique in size-based separation of proteins, playing a vital role in the biopharmaceutical industry for the analysis and characterization of therapeutic proteins, employing both UV and fluorescent detection. Understanding the effect of the operational parameters using easily applicable in migratio fluorescent labeling is increasingly critical, especially because multicapillary electrophoresis systems with fluorescent detection have recently gained prominence in high-throughput biopolymer analysis. In this study, the effects of the three most important user-adjustable operational parameters (temperature, gel concentration, and electric field strength) were investigated on the electrophoretic mobility and resolution of SDS-protein complexes in the presence of propidium iodide in the gel-buffer system. In the separation temperature study, the Arrhenius equation was applied to calculate the required activation energy for the electromigration through the propidium iodide-containing separation matrix for the sample proteins. With increasing gel concentration, the presence of the fluorophore in the gel-buffer system resulted in linear Ferguson plots, suggesting more predictable and consistent sieving behavior, in contrast to the non-linear plots observed earlier without the addition of the fluorescent dye. Finally, increase in the applied electric field resulted in elevated electrophoretic mobilities, both in the presence and absence of propidium ions in the sieving matrix. The resolution between the consecutively migrating SDS-protein complexes, on the other hand, decreased above 500 V/cm, probably due to conformation changes caused by the high field strengths. Our results underline the importance of optimizing these key parameters in SDS-CGE with propidium-mediated LIF detection to obtain rapid and high-resolution separation of complex protein samples including biopharmaceuticals.
十二烷基硫酸钠毛细管凝胶电泳（SDS-CGE）是一种常用于基于大小分离蛋白质的分析技术，在生物制药行业中，对于治疗性蛋白质的分析和表征起着至关重要的作用，它同时采用紫外和荧光检测。了解使用易于应用的荧光标记时操作参数的影响变得越来越关键，特别是因为带有荧光检测的多毛细管电泳系统最近在高通量生物聚合物分析中备受瞩目。在本研究中，研究了三个最重要的用户可调节操作参数（温度、凝胶浓度和电场强度）对凝胶缓冲系统中存在碘化丙啶时SDS-蛋白质复合物的电泳迁移率和分辨率的影响。在分离温度研究中，应用Arrhenius方程计算样品蛋白质通过含碘化丙啶的分离基质进行电迁移所需的活化能。随着凝胶浓度的增加，凝胶缓冲系统中荧光团的存在导致线性Ferguson图，这表明筛分行为更可预测且更一致，这与早期未添加荧光染料时观察到的非线性图形成对比。最后，施加电场的增加导致电泳迁移率升高，无论筛分基质中是否存在碘化丙啶离子。另一方面，连续迁移的SDS-蛋白质复合物之间的分辨率在高于500 V/cm时降低，这可能是由于高场强引起的构象变化。我们的结果强调了在使用碘化丙啶介导的激光诱导荧光检测的SDS-CGE中优化这些关键参数对于快速、高分辨率分离包括生物制药在内的复杂蛋白质样品的重要性。
