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

Yu Kyong Shin, Hyoung Joo Lee, Joon Seok Lee, Young Ki Paik

Research output: Contribution to journalArticlepeer-review

37 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1143-1150
Number of pages8
JournalProteomics
Volume6
Issue number4
DOIs
Publication statusPublished - 2006 Feb

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

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