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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.