Capillary Electrophoretic Methods for Protein and Microorganism Separations

Capillary electrophoresis (CE) is an analytical technique for the separation of molecules in a capillary under an applied electric field. CE generates high resolution separations in relatively short analysis times, with low reagent, and sample volume requirements. Two of the most common modes of CE are capillary zone electrophoresis (CZE) and capillary isoelectric focusing (cIEF). CZE separates analytes based on their size-to-charge ratios. It is the most common mode of CE due to its ability to separate molecules with high resolution and relative simplicity. cIEF is an orthogonal technique that separates analytes based on their isoelectric point (pI), which is the pH at which the average net charge of the molecule is zero. CZE and cIEF offer distinct advantages for high resolution separations of molecules in numerous fields including pharmaceutical, proteomic, genomic, and microbial analysis. CE instrumentation typically employs optical detection methods. However, optical detectors limit the information that you can obtain about the analytes. In this work, I improve instrumentation and methods for CE separations with two alternative detection schemes. (1) I developed an in-line cIEF interface to remove the concentration of ampholytes required for cIEF prior to MS detection. With the interface, an over 90% increase in area under the curve protein signal was observed. (2) I developed methods that enabled separation and collection of intact microorganisms for subsequent offline detection and analyses of cultured and uncultured species. These data represent significant improvement vs. existing methods. These efforts highlight the utility of CE for a diverse range of analytes with enhanced sensitivity and characterization.

毛细管电泳（Capillary electrophoresis, CE）是一种在施加电场下于毛细管内实现分子分离的分析技术。CE可在相对较短的分析时长内实现高分辨分离，且试剂与样品体积需求量较低。CE最常见的两种模式为毛细管区带电泳（Capillary zone electrophoresis, CZE）与毛细管等电聚焦（Capillary isoelectric focusing, cIEF）。CZE依据分析物的尺寸-电荷比实现分离，因其可实现高分辨分子分离且操作相对简便，成为最常用的CE模式。cIEF则是一种正交分析技术，依据分析物的等电点（isoelectric point, pI，即分子平均净电荷为零时的pH值）完成分离。CZE与cIEF在药物学、蛋白质组学、基因组学及微生物学分析等诸多领域的高分辨分子分离中各具独特优势。CE仪器通常采用光学检测方法，但这类光学检测器会限制可获取的分析物信息。本研究针对CE分离技术优化了仪器与方法，并提出两种替代检测方案：（1）开发了在线cIEF接口，可省去质谱（MS）检测前cIEF所需的两性电解质富集步骤，采用该接口后蛋白质信号的曲线下面积提升超过90%；（2）建立了可分离并收集完整微生物的方法，用于后续对可培养与不可培养物种的离线检测与分析。相较于现有方法，本研究的数据实现了显著改进。上述工作凸显了CE在多样分析物应用中的潜力，同时提升了检测灵敏度与表征能力。