Department of Chemical Engineering and Industrial Chemistry, Vrije Universiteit Brussel, 1050 Brussels, Belgium.
Novo Nordisk A/S, Global Research Technologies, DK-2760 Maaloev, Denmark.
Anal Chem. 2024 Aug 6;96(31):12827-12837. doi: 10.1021/acs.analchem.4c02441. Epub 2024 Jul 29.
Capillary isoelectric focusing (CIEF) coupled with electrospray ionization mass spectrometry (ESI-MS) is regarded as an outstanding approach for protein and proteoform analysis, combining a high-resolution separation technique and an enhanced detection technique. The few so-far developed CIEF-ESI-MS approaches exhibit limitations regarding sensitivity and separation performance. Here, we report a new generic methodology for CIEF-ESI-MS based on chemical mobilization, leading to highly efficient separation. This new integrated methodology relies on exchanging catholyte, initially introduced in the nanoCEasy interface in the focusing step, with sheath liquid (SL) in order to chemically mobilize the analytes into the ESI-MS system. The CIEF-MS method is evaluated by separation of a peptide set, model proteins, and monoclonal antibody charge variants. The effect of various parameters including master mixture composition, field strength, catholyte, SL composition, focusing time, and capillary conditions is optimized and discussed. Excellent separation performance can be achieved with a pI resolution down to 0.1 pH unit. The mobilization reproducibility is demonstrated with "migration time" RSDs below 10%. Additionally, the chemical mobilization is compared with the pressure assistance-chemical mobilization method, demonstrating that even a small pressure causes a strong decrease in separation performance, which clearly indicates the benefit of the chemical mobilization-based method. The applicability and separation power of the developed method are further exhibited by separation of Fc-conjugated insulins (mass = 62 kDa) differing in only one amino acid.
毛细管等电聚焦（CIEF）与电喷雾电离质谱（ESI-MS）结合被认为是一种出色的蛋白质和蛋白质形式分析方法，它结合了高分辨率分离技术和增强的检测技术。到目前为止，少数开发的 CIEF-ESI-MS 方法在灵敏度和分离性能方面存在局限性。在这里，我们报告了一种基于化学迁移的新型 CIEF-ESI-MS 通用方法，可实现高效分离。这种新的集成方法依赖于交换阴极液，最初在纳米 CEasy 接口中的聚焦步骤中引入，然后用鞘液 (SL) 进行化学迁移，将分析物转移到 ESI-MS 系统中。通过分离肽集、模型蛋白和单克隆抗体电荷变体来评估 CIEF-MS 方法。优化和讨论了各种参数的影响，包括主混合物组成、场强、阴极液、SL 组成、聚焦时间和毛细管条件。可以实现优异的分离性能，pI 分辨率低至 0.1 pH 单位。通过“迁移时间”RSD 低于 10%，证明了迁移的重现性。此外，将化学迁移与压力辅助-化学迁移方法进行了比较，结果表明，即使施加很小的压力也会导致分离性能强烈下降，这清楚地表明了基于化学迁移的方法的优势。通过分离仅在一个氨基酸上有所不同的 Fc 缀合胰岛素（质量=62 kDa），进一步展示了所开发方法的适用性和分离能力。
