Identification of differentially expressed proteins of gamma-ray irradiated rat intestinal epithelial IEC-6 cells by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry.

To identify proteins involved in the processes of cellular and molecular response to radiation damage repair in intestinal epithelial IEC-6 cells, we comparatively analyzed the proteome of irradiated IEC-6 cells with that of normal cells. A series of methods were used, including two-dimensional gel electrophoresis (Z-DE), PDQuest software analysis of 2-DE gels, peptide mass fingerprinting based on matrix-assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF-MS), and Swiss-Prot database searching, to separate and identify differentially expressed proteins. Western blotting and reverse transcriptase polymerase chain reaction (RT-PCR) were used to validate the differentially expressed proteins. Image analysis revealed that averages of 608 +/- 39 and 595 +/- 31 protein spots were detected in normal and irradiated IEC-6 cells, respectively. Sixteen differential protein spots were isolated from gels, and measured with MALDI-TOF-MS. A total of 14 spots yielded good spectra, and 11 spots matched with known proteins after database searching. These proteins were mainly involved in anti-oxidation, metabolism, and protein post-translational processes. Western blotting confirmed that stress-70 protein was down-regulated by gamma-irradiation. Up-regulation of ERP29 was confirmed by RT-PCR, indicating that it is involved in ionizing radiation. The clues provided by the comparative proteome strategy utilized here will shed light on molecular mechanisms of radiation damage repair in intestinal epithelial cells.