Wagner K, Racaityte K, Unger K K, Miliotis T, Edholm L E, Bischoff R, Marko-Varga G
Institut für Anorganische Chemie und Analytische Chemie, Johannes Gutenberg-Universität, Mainz, Germany.
J Chromatogr A. 2000 Oct 6;893(2):293-305. doi: 10.1016/s0021-9673(00)00736-6.
Current developments in drug discovery in the pharmaceutical industry require highly efficient analytical systems for protein mapping providing high resolution, robustness, sensitivity, reproducibility and a high throughput of samples. The potential of two-dimensional (2D) HPLC as a complementary method 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 chromatography in the first dimension and reversed-phase chromatography in the second dimension. We used a comprehensive two-dimensional approach based on different separation speeds. In the first dimension 2.5 microm 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 dimension, short columns of 14 x 4.6 mm I.D. with 1.5 microm n-octadecyl bonded, non-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 dimension. The analyte fractions from the ion-exchange column were transferred alternatively to one of the two reversed-phase columns using a 10-port switching 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 cases, be baseline-separated in the second dimension. The total value of peak capacity was calculated to 600. Fully unattended overnight runs for repeatability studies proved the applicability of the system. The values for the relative standard deviation (RSD) of the retention times of proteins were less 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-resolution protein separations with a total analysis time of less than 20 min, equivalent to the run time of the first dimension.
制药行业药物研发的当前进展需要高效的蛋白质图谱分析系统，该系统要具备高分辨率、稳健性、灵敏度、可重复性以及高通量样本处理能力。研究了二维（2D）高效液相色谱作为二维凝胶电泳补充方法的潜力，特别是在速度和重复性方面。该方法将应用于分子量小于20000的蛋白质，这些蛋白质在二维凝胶电泳中分辨率不佳。本文所述的二维高效液相色谱系统由第一维的阴离子或阳离子交换色谱和第二维的反相色谱组成。我们采用了基于不同分离速度的全面二维方法。在第一维中，分别键合二乙氨基乙基和磺酸基团的2.5微米聚合物珠作为离子交换剂，流速为1毫升/分钟。为了在第二维中实现非常高速和高分辨率的分离，选择了内径为14×4.6毫米、填充1.5微米正十八烷基键合无孔硅胶的短柱，流速为2.5毫升/分钟。在第二维中并行使用两根反相柱。来自离子交换柱的分析物馏分使用10通切换阀交替转移到两根反相柱中的一根。分析物以柱上聚焦模式沉积在一根柱的顶部，而第二根柱上的分析物被洗脱。在大多数情况下，在第一维中未完全分离的蛋白质在第二维中可以实现基线分离。计算得到的总峰容量值为600。用于重复性研究的完全无人值守的过夜运行证明了该系统的适用性。蛋白质保留时间的相对标准偏差（RSD）值小于1%（n = 15），而峰面积的RSD平均小于15%（n = 15）。平均检测限为300纳克蛋白质，对于卵清蛋白则降至50纳克。二维高效液相色谱系统提供了高分辨率的蛋白质分离，总分析时间不到20分钟，与第一维的运行时间相当。
