Department of Chemistry, Aalen University, Aalen, Germany.
Electrophoresis. 2012 May;33(9-10):1356-66. doi: 10.1002/elps.201100620.
Capillary electrophoresis-mass spectrometry (CE-MS) more and more gains in importance as an analytical technique for the identification and characterization of intact proteins in the biopharmaceutical area. Thus, a CE-ESI-MS method was optimized and validated systematically with respect to the improved screening and characterization of intact proteins. The optimization was accomplished by variation of different CE-MS parameters, such as capillary coating, background electrolyte, sheath liquid, and nebulizer gas pressure, while monitoring both the resolution and signal intensities. Achievable separation is discussed quantitatively in the context of the coating and the resulting EOF, the protein mobilities, and the suction effect of the sprayer. The observed precisions of the optimized method regarding the migration times (mean RSD = 1.4%) and peak areas (mean RSD = 12.3%) and an extensive principal component analysis revealed that the presented method is reliable and useful for the quantitation of intact proteins and protein isoforms. The applicability of this method to various proteins showing different characteristics (pI value, molecular mass, hydrophobicity, etc.) is discussed. The presented method will contribute to the improved characterization of a large variety of intact proteins in the biomedical and pharmaceutical area.
毛细管电泳-质谱联用（CE-MS）作为一种分析技术，在生物制药领域中越来越重要，用于鉴定和表征完整蛋白质。因此，我们系统地优化和验证了 CE-ESI-MS 方法，以提高对完整蛋白质的筛选和表征能力。通过改变不同的 CE-MS 参数（如毛细管涂层、背景电解质、鞘液和雾化器气体压力）来实现优化，同时监测分辨率和信号强度。在讨论可实现的分离时，我们考虑了涂层和由此产生的EOF、蛋白质迁移率以及喷雾器的抽吸效应的定量影响。优化方法在迁移时间（平均 RSD=1.4%）和峰面积（平均 RSD=12.3%）方面的观察精度以及广泛的主成分分析表明，该方法可靠且可用于定量分析完整蛋白质和蛋白质异构体。讨论了该方法对具有不同特性（等电点值、分子量、疏水性等）的各种蛋白质的适用性。该方法将有助于改善生物医学和制药领域中各种完整蛋白质的表征。
