Department of Chemistry and Biochemistry, University of Oklahoma, USA.
Department of Microbiology and Plant Biology, University of Oklahoma, USA.
Talanta. 2018 May 15;182:225-229. doi: 10.1016/j.talanta.2018.01.072. Epub 2018 Feb 1.
A comprehensive two-dimensional liquid chromatography (LCxLC) system consisting of twelve columns in the second dimension was developed for comprehensive analysis of intact proteins in complex biological samples. The system consisted of an ion-exchange column in the first dimension and the twelve reverse-phase columns in the second dimension; all thirteen columns were monolithic and prepared inside 250 µm i.d. capillaries. These columns were assembled together through the use of three valves and an innovative configuration. The effluent from the first dimension was continuously fractionated and sequentially transferred into the twelve second-dimension columns, while the second-dimension separations were carried out in a series of batches (six columns per batch). This LCxLC system was tested first using standard proteins followed by real-world samples from E. coli. Baseline separation was observed for eleven standard proteins and hundreds of peaks were observed for the real-world sample analysis. Two-dimensional liquid chromatography, often considered as an effective tool for mapping proteins, is seen as laborious and time-consuming when configured offline. Our online LCxLC system with increased second-dimension columns promises to provide a solution to overcome these hindrances.
建立了一个由十二根二维柱组成的二维液相色谱（LCxLC）系统，用于对复杂生物样品中的完整蛋白质进行全面分析。该系统由第一维的离子交换柱和第二维的十二根反相柱组成；所有十三根柱均为整体式，在 250 µm 内径的毛细管内制备。这些柱子通过三个阀和一种创新的配置组装在一起。第一维的流出物连续进行分段，并顺序进入十二根二维柱，而二维分离则在一系列批次中进行（每批六个柱）。该 LCxLC 系统首先使用标准蛋白质进行了测试，然后使用来自大肠杆菌的实际样品进行了测试。观察到十一种标准蛋白质的基线分离，并观察到实际样品分析的数百个峰。二维液相色谱通常被认为是一种用于蛋白质图谱的有效工具，但当离线配置时，它被认为是繁琐和耗时的。我们的在线 LCxLC 系统增加了二维柱，有望提供一种解决方案来克服这些障碍。
