Proteomic analysis of mammalian basic proteins by liquid‐based two‐dimensional column chromatography

To develop a standard method for separating highly basic proteins in mammalian cells, we established a 2‐D LC separation system coupled with chromatofocusing/nonporous RP column chromatography (CF/NPRPC) in a ProteomeLab PF2D system. After standardizing conditions for 2‐D LC, a 2‐D liquid protein map of uninfected macrophage proteins with pH range 8.3–11.3 was constructed, and then compared with a macrophage protein map made after infection with Candida albicans . The results demonstrate that 2‐D LC offers both high resolution and reproducibility for separation of highly basic, macrophage proteins. After protein identification using a nano 2‐D LC‐MS/MS Proteomics Solution System, quantitative determination of the changes in the differentially expressed proteins ( e.g., galectin‐3) in C. albicans ‐infected macrophages was also accomplished by measuring the peak area of the chromatogram in 2‐D LC. The result from this measurement of galectin‐3 expression shows a 3.41‐fold decrease in the infected macrophage cells, which was further confirmed by that from the RT‐PCR of mRNA of galectin‐3. Thus, 2‐D LC coupled with CF/NPRPC could be applicable to common analysis of highly basic proteins in a high‐throughput manner.