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

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.

SUBMITTER: Zhang B

PROVIDER: S-EPMC4612041 | biostudies-literature | 2003 Dec

REPOSITORIES: biostudies-literature

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

Zhang Bo B Su Yong-Ping YP Ai Guo-Ping GP Liu Xiao-Hong XH Wang Feng-Chao FC Cheng Tian-Min TM

World journal of gastroenterology 20031201 12

<h4>Aim</h4>To identify the differentially expressed proteins involved in ionizing radiation in mice and to explore new ways for studying radiation-related proteins.<h4>Methods</h4>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- ...[more]

PMID: 14669322

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Project description:A method of analysis is presented that utilizes matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to monitor the kinetics and products of RNA cleavage, by use of a program designed to mass-match observed MS peaks with predicted RNA cleavage products. The method is illustrated through application to the study of targeted oxidation of RNA stem loops from HIV-1 Rev Response Element mRNA (RRE RNA) and ribosomal 16S A-site RNA (16S RNA) by metallonucleases. Following incubation of each RNA with catalysts and/or redox co-reactants, reaction mixtures were desalted, and MALDI-TOF MS was used to monitor both time-resolved formation of cleavage products and disappearance of full-length RNA. For each RNA, a unique list was generated that contained the predicted masses of both the full-length, and all of the possible RNA cleavage fragments that resulted from the combination of all possible cleavage sites and each of the six expected overhangs formed at nascent termini adjacent to the cleavage sites. The overhangs corresponded to 2',3'-cyclic phosphate, 3'-phosphate, 3'-phosphoglycolate, 5'- hydroxyl and 5'- phosphate, which corresponded to differing oxidative, hydrolytic, and/or 2'-OH-mediated-endonucleolytic modes of scission. Each mass spectrum was compared with a corresponding list of predicted masses, and peaks were rapidly assigned by use of a Perl script, with a mass-matching tolerance of 200 ppm. Both time-dependent cleavage mediated by metallonucleases and MALDI-TOF-induced fragmentation were observed, and these were distinguished by time-dependent experiments. The resulting data allowed a semi-quantitative assessment of the rate of formation of each overhang at each nucleotide position. Limitations included artifactual skewing of quantification by mass bias, a limited mass range for quantification, and a lack of detection of secondary cleavage products. Nevertheless, the method presented herein provides a rapid, accurate, highly-detailed and semi-quantitative analysis of RNA cleavage that should be widely applicable.

Dataset Information

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

Publications

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

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