Technische Universität Braunschweig, Institute of Medicinal and Pharmaceutical Chemistry, Beethovenstraße 55, 38106, Braunschweig, Germany.
Technische Universität Braunschweig, Institute of Pharmacology, Toxicology and Clinical Pharmacy, Mendelssohnstraße 1, 38106, Braunschweig, Germany.
Anal Bioanal Chem. 2022 Feb;414(4):1699-1712. doi: 10.1007/s00216-021-03797-4. Epub 2021 Dec 6.
Two-dimensional separations provide a simple way to increase the resolution and peak capacity of complex protein separations. The feasibility of a recently developed instrumental approach for two-dimensional separations of proteins was evaluated. The approach is based on the general principle of two-dimensional gel electrophoresis. In the first dimension, semi-preparative strong anion exchange high-performance liquid chromatography is utilized and fractions are collected by means of a fraction collector. They are subsequently analyzed in the second dimension with microchip capillary electrophoresis sodium dodecyl sulfate. Microchip capillary electrophoresis provides the necessary speed (approximately 1 min/fraction) for short analysis. In this study, three different samples were investigated. Different constructs of soluble guanylyl cyclase were expressed in Sf9-cells using the baculovirus expression system. Cell lysates were analyzed and the resulting separations were compared. In our experimental setup, the soluble guanylyl cyclase was identified among hundreds of other proteins in these cell lysates, indicating its potential for screening, process control, or analysis. The results were validated by immunoblotting. Samples from Chinese hamster ovary cell culture before and after a purification step were investigated and approximately 9% less impurities could be observed. The separation patterns obtained for human plasma are closely similar to patterns obtained with two-dimensional gel electrophoresis and a total of 218 peaks could be observed. Overall, the approach was well applicable to all samples and, based on these results, further directions for improvements were identified. .
二维分离提供了一种增加复杂蛋白质分离分辨率和峰容量的简单方法。评估了一种最近开发的用于蛋白质二维分离的仪器方法的可行性。该方法基于二维凝胶电泳的一般原理。在第一维中，使用半制备强阴离子交换高效液相色谱法，并通过馏分收集器收集馏分。然后，它们在第二维中用微芯片毛细管电泳十二烷基硫酸钠进行分析。微芯片毛细管电泳提供了进行短分析所需的速度（约 1 分钟/馏分）。在这项研究中，研究了三个不同的样本。使用杆状病毒表达系统在 Sf9 细胞中表达了不同结构的可溶性鸟苷酸环化酶。分析细胞裂解物并比较得到的分离结果。在我们的实验设置中，在这些细胞裂解物中，从数百种其他蛋白质中鉴定出可溶性鸟苷酸环化酶，表明其在筛选、过程控制或分析方面具有潜力。结果通过免疫印迹进行了验证。研究了经过纯化步骤前后的中国仓鼠卵巢细胞培养物的样品，可观察到大约 9%的杂质减少。获得的人血浆分离模式与二维凝胶电泳获得的模式非常相似，总共可以观察到 218 个峰。总体而言，该方法适用于所有样品，并且根据这些结果，确定了进一步改进的方向。
