Two−dimensional separation of human plasma proteins using agarose gel isoelectric focusing followed by SDS capillary electrophoresis

Two−dimensional polyacryiamide gel electrophoresis (2−D PAGE) has been widely used for protein separation because of its high resolution; more than one thousand proteins or polypeptides can be separated according to the differences of their isoelectric point (pI) and molecular mass. However, 2−D PAGE involves many steps of manual handling, sample injection, setting gel molds in an electrophoresis apparatus, removal gels from the molds, staining and destaining, etc. For quantitative analysis of protein on stained gels, more steps of manual handling are required in computer−assisted image analysis, densitometry [1−3]. On the other hand, there are non−gel separation methods of proteins such as HPLC and capillary electrophoresis (CE). Although these methods have an advantage that they can be automated, it is difficult to separate complex protein mixtures into individual protein peaks or fractions employing only one of these separation methods [3]. Therefore, multi dimensional separation systems have been reported for protein analysis, employing a non−gel separation method at the last dimension of the separation [4−8]. However, it is preferable to combine a method which separate proteins according to pI differences with the one which separate according to molecular mass in order to correlate the analysis results with the protein information accumulated during these 25 years using 2−D PAGE. We have established the conditions of SDS−CE, using linear polyacrylamide (LPA) as a sieving matrix, to separate human plasma proteins according to their size differences [9]. About 30 UV (230 nm) absorbing peaks and shoulders were detected within 60 min when fused silica capillaries of 30 cm effective length and an automated CE apparatus were employed. These results showed that this method is suited as the last step of multi−dimensional separation of proteins. In this paper, we report on two−dimensional separation of human plasma proteins combining agarose gel isoelectric focusing (IEF) with SDS−CE. Proteins were separated under non−denaturing conditions in an agarose gel IEF column, the gel was dissected into 32 slices, then the proteins were extracted from the gel slices and subjected to SDS−CE. The multiple CE patOriginal