Differentially expressed proteins of gamma-ray irradiated mouse intestinal epithelial cells by two-dimensional electrophoresis and MALDI-TOF mass spectrometry.
OBJECTIVE
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.


CONCLUSIONS
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.
Zhang
Bo
B
Institute of Combined Injury of PLA, Third Military Medical University, Chongqing 400038, China.
Su
Yong-Ping
YP
Ai
Guo-Ping
GP
Liu
Xiao-Hong
XH
Wang
Feng-Chao
FC
Cheng
Tian-Min
TM
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D015180:Electrophoresis, Gel, Two-Dimensional; D004798:Enzymes / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D005720:Gamma Rays; D007091:Image Processing, Computer-Assisted; D007413:Intestinal Mucosa / Q000166:cytology / Q000502:physiology / Q000528:radiation effects*; D051379:Mice; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017931:DNA Primers; D004798:Enzymes; D011506:Proteins