Protein mapping by two-dimensional high performance liquid chromatography

Current developments in drug discovery in the pharmaceutical industry requi re highly efficient analytical systems for protein mapping providing high r esolution, robustness, sensitivity, reproducibility and a high throughput o f samples. The potential of two-dimensional (2D) HPLC as a complementary me thod to 2D-gel electrophoresis is investigated, especially in view of speed and repeatability. The method will be applied for proteins of a molecular mass <20 000 which are not well resolved in 2D-gel electrophoresis. The 2D- HPLC system described in this work consisted of anion- or cation-exchange c hromatography in the first dimension and reversed-phase chromatography in t he second dimension. We used a comprehensive two-dimensional approach based on different separation speeds. In the first dimension 2.5 mu m polymeric beads bonded with diethylaminoethyl and sulfonic acid groups, respectively, were applied as ion exchangers and operated at a flow-rate of 1 ml/min. To achieve very high-speed and high-resolution separations in the second dime nsion, short columns of 14x4.6 mm I.D. with 1.5 mu m n-octadecyl bonded, no n-porous silica packings were chosen and operated at a flow-rate of 2.5 ml/ min. Two reversed-phase columns were used in parallel in the second dimensi on. The analyte fractions from the ion-exchange column were transferred alt ernatively to one of the two reversed-phase columns using a 10-port switchi ng valve. The analytes were deposited in an on-column focusing mode on top of one column while the analytes on the second column were eluted. Proteins , which were not completely resolved in the first dimension can, in most ca ses, be baseline-separated in the second dimension. The total value of peak capacity was calculated to 600. Fully unattended overnight runs for repeat ability studies proved the applicability of the system. The values for the relative standard deviation (RSD) of the retention times of proteins were l ess than 1% (n=15), while the RSDs of the peak areas were less than 15% (n= 15) on average. The limit of detection was 300 ng of protein on average and decreased to 50 ng for ovalbumin. The 2D-HPLC system offered high-resoluti on protein separations with a total analysis time of less than 20 min, equi valent to the run time of the first dimension. (C) 2000 Published by Elsevi er Science B.V.