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Proteomic analysis of mammalian basic proteins by liquid‐based two‐dimensional column chromatography
Author(s) -
Shin YuKyong,
Lee HyoungJoo,
Lee Joon Seok,
Paik YoungKi
Publication year - 2006
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200500433
Subject(s) - chromatofocusing , chromatography , proteomics , candida albicans , chemistry , proteome , biology , biochemistry , size exclusion chromatography , enzyme , genetics , gene
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.