School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Bd d'Yvoy 20, 1211 Geneva 4, Switzerland.
School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Bd d'Yvoy 20, 1211 Geneva 4, Switzerland.
Anal Chim Acta. 2014 Feb 27;813:97-105. doi: 10.1016/j.aca.2013.12.043. Epub 2014 Jan 10.
Capillary electrophoresis (CE) coupled with electrospray ionization (ESI) mass spectrometry (MS) is a suitable technique for the analysis of intact proteins. The main configuration to realize this coupling is the sheath liquid interface, which is characterized by the addition of a make-up liquid providing the electric contact as well as the appropriate flow and solvent composition for optimal ionization and evaporation. One main advantage of this interface is that the composition of the sheath liquid can be tuned to modify the ionization without affecting CE selectivity and efficiency. In the case of protein ionization, this feature is particularly interesting to modulate their charge-state distribution (CSD), while keeping the separation performance unchanged. In this context, the current work evaluated the effect on proteins' CSD of adding supercharging molecules to the sheath liquid. Several supercharging reagents were tested with different background electrolyte (BGE) and their impact was estimated for three model proteins (i.e., human insulin, human growth hormone, hemoglobin A0) exhibiting various properties in terms of ionization, conformation, and flexibility. Their influence on the global sensitivity for each protein was also assessed. Among the supercharging reagents tested, m-NBA and sulfolane led to supercharging effect whose magnitude depended on the proteins as well of the BGE pH. The sensitivity and separation performance remained globally unchanged for each protein and supercharging additive, while sulfolane led in some cases to a sensitivity improvement.
毛细管电泳（CE）与电喷雾电离（ESI）质谱（MS）联用是分析完整蛋白质的合适技术。实现这种耦合的主要配置是鞘液界面，其特点是添加一种补加液，提供电接触以及适当的流动和溶剂组成，以实现最佳的离子化和蒸发。该接口的一个主要优点是，可以调整鞘液的组成来调节离子化，而不会影响 CE 的选择性和效率。在蛋白质离子化的情况下，这种特性特别有趣，可以调节其电荷状态分布（CSD），同时保持分离性能不变。在这种情况下，当前的工作评估了向鞘液中添加超荷分子对蛋白质 CSD 的影响。用不同的背景电解质（BGE）测试了几种超荷试剂，并评估了它们对三种模型蛋白质（即人胰岛素、人生长激素、血红蛋白 A0）的影响，这些蛋白质在离子化、构象和灵活性方面具有不同的特性。还评估了它们对每种蛋白质整体灵敏度的影响。在所测试的超荷试剂中，m-NBA 和环丁砜导致了超荷效应，其大小取决于蛋白质以及 BGE 的 pH 值。每种蛋白质和超荷添加剂的灵敏度和分离性能总体上保持不变，而在某些情况下，环丁砜会提高灵敏度。
