Getting the Best out of Capillary Electrophoresis and Capillary Electrophoresis–Mass Spectrometry by Quantifying Sources of Peak Broadening for Proteins Using Polyelectrolyte Multilayer Coated Fused Silica Capillaries,Analytical Chemistry

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Getting the Best out of Capillary Electrophoresis and Capillary Electrophoresis–Mass Spectrometry by Quantifying Sources of Peak Broadening for Proteins Using Polyelectrolyte Multilayer Coated Fused Silica Capillaries
Analytical Chemistry ( IF 6.7 ) Pub Date : 2024-09-10 , DOI: 10.1021/acs.analchem.4c02276
Laura Dhellemmes ₁ , Laurent Leclercq ₁ , Alisa Höchsmann _{2,

3} , Christian Neusüß ₂ , Michel Martin ₄ , Hervé Cottet ₁

Affiliation

Capillary electrophoresis (CE) has emerged as a relevant technique for protein and biopharmaceutical analysis, as it combines high separation efficiency, sensitivity, and versatility. The use of capillary coatings, including successive multiple ionic-polymer layers (SMILs), reduces interactions between analytes and the capillary, further improving the CE performance. Nevertheless, separations done on SMIL coatings rarely surpass 500 × 10³ plates/m. To obtain the best out of the CE, it is interesting to have a detailed look at the sources of peak dispersion. Separations of a mix of model proteins were performed on (poly(diallyldimethylammonium chloride)/poly(styrenesulfonate))_2.5-coated capillaries at different electrical field strengths, leading to plate height H against migration velocity u plots that enabled a quantitative analysis of each contribution. Using this model, capillary lengths and injected volumes were systematically varied. For the first time, the contribution of sample electrophoretic heterogeneity to the total peak dispersion was deciphered for model proteins and a monoclonal antibody. Dispersion due to electromigration was seen to have an impact on plate heights in the case of triangular peaks of small molecules but not for proteins under the present conditions. UV and mass spectrometry detections were compared on the same capillary, providing valuable information on the impact of the detection type on separation efficiency. Close to 1 million plates/m were reached in the best conditions.

中文翻译：

通过使用聚电解质多层涂层熔融石英毛细管定量蛋白质峰展宽的来源，充分利用毛细管电泳和毛细管电泳-质谱法

毛细管电泳 (CE) 已成为蛋白质和生物制药分析的相关技术，因为它结合了高分离效率、灵敏度和多功能性。使用毛细管涂层，包括连续的多个离子聚合物层 (SMIL)，可减少分析物与毛细管之间的相互作用，从而进一步提高 CE 性能。然而，SMIL 涂层上的分离很少超过 500 × 10 ³板/米。为了充分发挥 CE 的作用，详细研究峰色散的来源是很有趣的。在不同电场强度下的（聚（二烯丙基二甲基氯化铵）/聚（苯乙烯磺酸）） _2.5涂层毛细管上进行模型蛋白质混合物的分离，得到板高度H与迁移速度u 的关系图，从而能够对每种贡献进行定量分析。使用该模型，系统地改变毛细管长度和注射体积。首次破译了模型蛋白和单克隆抗体的样品电泳异质性对总峰分散的影响。在小分子的三角峰的情况下，由于电迁移引起的分散被认为对板高度有影响，但在当前条件下对蛋白质没有影响。在同一毛细管上比较紫外和质谱检测，提供有关检测类型对分离效率影响的有价值的信息。在最佳条件下达到了接近 100 万个板/米。

更新日期：2024-09-10

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