Optimization of two-dimensional off-line LC/MS separations to improve resolution of complex proteomic samples

In off-line 2D-HPLC a continuous salt gradient is applied in the first separation dimension. This increases the number of identified proteins from complex samples significantly due to higher chromatographic resolution compared to stepwise elution. Achievement of optimal resolution requires the optimization of the two separation dimensions. The influence of LC elution gradients in the first and second dimensions, of analysis time, of stationary-phase material, and of column dimensions was systematically investigated in order to obtain information on the overall peak capacity of the separation system. Provided data indicate that for complex samples such as an E. coli cell extract, a shallow LC SCX gradient with a high number of collected fractions significantly increases the overall peak capacity while for lower complexity samples short gradients with few fractions were sufficient to obtain a maximum of identified peptides. In addition, column dimensions and materials exhibited a strong effect on the overall efficiency of the 2D HPLC separation. The outcome of these experiments could hence serve as a guideline for investigators to adapt their method for the separation of their specific proteome sample to achieve a maximum of peptide sequence information by 2D LC MS/MS analysis.