Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 9 (12), 2726-31

Differentially Expressed Proteins of Gamma-Ray Irradiated Mouse Intestinal Epithelial Cells by Two-Dimensional Electrophoresis and MALDI-TOF Mass Spectrometry

Affiliations

Differentially Expressed Proteins of Gamma-Ray Irradiated Mouse Intestinal Epithelial Cells by Two-Dimensional Electrophoresis and MALDI-TOF Mass Spectrometry

Bo Zhang et al. World J Gastroenterol.

Abstract

Aim: To identify the differentially expressed proteins involved in ionizing radiation in mice and to explore new ways for studying radiation-related proteins.

Methods: Bal B/c mice grouped as sham-irradiation, 3 h and 72 h irradiation were exposed to 9.0 Gy single dose of gamma-irradiation. Intestinal epithelia were isolated from mice, and total proteins were extracted with urea containing solution. A series of methods were used, including two-dimensional electrophoresis, PDQuest 2-DE software analysis, peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and SWISS-PROT database searching, to separate and identify the differential proteins. Western blotting and RT-PCR were used to validate the differentially expressed proteins.

Results: Mouse intestine was severely damaged by 9.0 Gy gamma-irradiation. Image analysis of two-dimensional gels revealed that averages of 638 +/- 39, 566 +/- 32 and 591 +/- 29 protein spots were detected in 3 groups, respectively, and the majority of these protein spots were matched. About 360 protein spots were matched between normal group and 3 h irradiation group, and the correlation coefficient was 0.78 by correlation analysis of gels. Also 312 protein spots matched between normal group and 72 h irradiation group, and 282 protein spots between 3 h and 72 h irradiation groups. Twenty-eight differential protein spots were isolated from gels, digested with trypsin, and measured with MALDI-TOF-MS. A total of 25 spots yielded good spectra, and 19 spots matched known proteins after database searching. These proteins were mainly involved in anti-oxidation, metabolism, signal transduction, and protein post-translational processes. Western-blotting confirmed that enolase was up-regulated by gamma-irradiation. Up-regulation of peroxiredoxin I was verified by applying RT-PCR technique, but no change occurred in Q8VC72.

Conclusion: These differentially expressed proteins might play important roles when mouse intestine was severely injured by gamma-irradiation. It is suggested that differential proteomic analysis may be a useful tool to study the proteins involved in radiation damage of mouse intestinal epithelia.

Figures

Figure 1
Figure 1
Histological changes in crypts following irradiation. A: Unirradiated controls (×100). B: 3 hours after a single dose of 9.0 Gy (×100). Villi and crypts remained unchanged, but cell division of intestinal epithelia ceased. C: 3 days after a single dose of 9.0 Gy (×100).
Figure 2
Figure 2
Two-dimensional electrophoresis maps. A: normal intestinal epithelial cells, B: 3 h irradiated epithelial cells, C: 72 h post-radiation. Number indicated proteins were summarized in Table 2.
Figure 3
Figure 3
MALDI-TOF-MS spectrum of spot P35700 in 2-DE map. MS spectrum of peptide mixture was obtained from a typical in-gel digestion of the 2-DE separated protein.
Figure 4
Figure 4
Western blot analysis of Enolase after radiation. A: Protein samples, obtained at indicated time after radiation, were separated by SDS-PAGE, and immunoblotted with corresponding antibody. Equal amounts of total proteins were applied to each lane. B: Relative protein level of Enolase was determined by quantitating the intensity of Enolase bands with a densitometer.
Figure 5
Figure 5
Semi-quantitative RT-PCR analysis of peroxiredoxin I. A: PCR product run on an agarose gel. The upper band repre-sented peroxiredoxin I (750 bp), and the lower band represented GAPDH (450 bp). B: Relative abundance of the mRNA of peroxiredoxin I was normalized by GAPDH.
Figure 6
Figure 6
Semi-quantitative RT-PCR analysis of Q8VC72. RNA samples were prepared from mouse intestinal epithelia at indi-cated time. Left: The band represents Q8VC72 (461 bp). Right: The band represents GAPDH (450 bp). The expression of Q8VC72 remained unchanged after radiation.

Similar articles

See all similar articles

Cited by 7 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback