Abstract
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.Graphical abstract.
Highlights
The use of multi-dimensional separations for complex samples is a proven and tested strategy to facilitate an improved separation and easier analysis of complex samples, which are often insufficiently resolved using only one separation technique [1]
The ever-present peak at approximately 7 kDa is a system peak originating from sodium dodecyl sulfate (SDS) micelles
There are some variations in their intensity, which is in accordance with the expectations, as the
Summary
The use of multi-dimensional separations for complex samples is a proven and tested strategy to facilitate an improved separation and easier analysis of complex samples, which are often insufficiently resolved using only one separation technique [1]. As pointed out by Ranjbar et al [3], the popularity of HPLC × CE is much lower than HPLC × HPLC, despite the high orthogonality that can be achieved [3, 4, 6] Such combinations provide a unique selectivity and may offer an alternative to other approaches. Even though, both capillary electrophoresis sodium dodecyl sulfate (CE-SDS) and ion exchange chromatography (IEX) are frequently employed for the characterization of proteins [9,10,11,12], the coupling of both techniques has not received much attention [13]
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