Comprehensive two-dimensional separation in coupling of reversed-phase chromatography with capillary isoelectric focusing followed by MALDI-MS identification using on-target digestion for intact protein analysis

A coupling of capillary RP LC as the first dimension with CIEF as the second dimension followed by MALDI-MS identification was demonstrated. Based on 2-D separation system developed by our group (Electrophoresis 2003, 24, 3289-3295), this paper focused on incorporating tryptic digestion into the top-down proteomics methodology, retaining the benefits of the top-down method. Hydrophobic layer of packing-material C18 coated with SE-30 on the MALDI-target surface was used to permit the CIEF fractions to be easily concentrated and free of ampholytes using on-target washing. Following the removal of ampholytes, on-target tryptic digestion was performed to generate PMF for protein identification. Using the proteome analytical strategy, we could obtain not only intact protein p/ value but also PMF for accurate protein identification. The feasibility of the strategy was first tested with a mixture of model proteins with different p/s and molecular masses. Proteome of rat liver tissue extracts was further analyzed using the system for verification. The results have shown that the system is effective for complex proteomic analysis.