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Proteomic profiling of aging in glomeruli of mice by using two-dimensional differential gel electrophoresis.

ABSTRACT: BACKGROUND: Glomerular proteins were analyzed by proteomics to screen proteins participating in maturation of glomeruli before senescence and to find key proteins involved in the aging process. MATERIAL AND METHODS: Glomeruli of C57BL/6 mice at 8 and 20 weeks were separated by kidney perfusion. Proteomic profiles of glomeruli were investigated by using two-dimensional differential gel electrophoresis and MALDI-TOF mass spectrometry. RESULTS: We identified 22 differentially expressed proteins. Among them, 3 proteins were significantly up-regulated and 19 proteins were significantly down-regulated in mature mice. Out of these 22 proteins, 18% take part in protein transport, protein targeting, and proteolysis; 5% in glycolysis; 14% in transcription; 9% in electron transport; 9% were chaperones; and 9% were hydrolases. CONCLUSIONS: Our results provide insights into the glomerular differentially expressed proteins correlated with renal aging. In this study we found that aging altered the expression of ATP synthase subunit beta. Further studies on this protein might help to understand the mechanism of renal aging.

SUBMITTER: Liu X

PROVIDER: S-EPMC4548697 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Proteomic profiling of aging in glomeruli of mice by using two-dimensional differential gel electrophoresis.

Liu Xiaodan X Fan Qiuling Q Yang Gang G Wang Lining L

Medical science monitor : international medical journal of experimental and clinical research 20150207

<h4>Background</h4>Glomerular proteins were analyzed by proteomics to screen proteins participating in maturation of glomeruli before senescence and to find key proteins involved in the aging process.<h4>Material and methods</h4>Glomeruli of C57BL/6 mice at 8 and 20 weeks were separated by kidney perfusion. Proteomic profiles of glomeruli were investigated by using two-dimensional differential gel electrophoresis and MALDI-TOF mass spectrometry.<h4>Results</h4>We identified 22 differentially exp ...[more]

PMID: 25659849

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Project description:BACKGROUND:Proteome analysis is frequently applied in identifying the proteins or biomarkers in knee synovial fluids (SF) that are associated with osteoarthritis and other arthritic disorders. The 2-dimensional gel electrophoresis (2-DE) is the technique of choice in these studies. Disease biomarkers usually appear in low concentrations and may be masked by high abundant proteins. Therefore, the main aim of this study was to find the most suitable sample preparation method that can optimize the expression of proteins on 2-DE gels that can be used to develop a reference proteome picture for non-osteoarthritic knee synovial fluid samples. Proteome pictures obtained from osteoarthritic knee synovial fluids can then be compared with the reference proteome pictures obtained in this study to assist us in identifying the disease biomarkers more correctly. RESULTS:The proteomic tool of 2-DE with immobilized pH gradients was applied in this study. A total of 12 2-DE gel images were constructed from SF samples that were free of osteoarthritis. In these samples, 3 were not treated with any sample preparation methods, 3 were treated with acetone, 3 were treated with 2-DE Clean-Up Kit, and 3 were treated with the combination of acetone and 2-D Clean-Up Kit prior to 2-DE analysis. Gel images were analyzed using the PDQuest Basic 8.0.1 Analytical software. Protein spots that were of interest were excised from the gels and sent for identification by mass spectrometry. Total SF total protein concentration was calculated to be 21.98 ± 0.86 mg/mL. The untreated SF samples were detected to have 456 ± 33 protein spots on 2-DE gel images. Acetone treated SF samples were detected to have 320 ± 28 protein spots, 2-D Clean-Up Kit treated SF samples were detected to have 413 ± 31 protein spots, and the combined treatment method of acetone and 2-D Clean-Up Kit was detected to have 278 ± 26 protein spots 2-DE gel images. SF samples treated with 2-D Clean-Up Kit revealed clearer presentation of the isoforms and increased intensities of the less abundant proteins of haptoglobin, apolipoprotein A-IV, prostaglandin-D synthase, alpha-1B-glycoprotein, and alpha-2-HS-glycoprotein on 2-DE gel images as compared with untreated SF samples and SF samples treated with acetone. CONCLUSIONS:The acetone precipitation method and the combined treatment effect of acetone and 2-DE Clean-Up Kit are not preferred in preparing SF samples for 2-DE analysis as both protein intensities and numbers decrease significantly. On the other hand, 2-D Clean-Up Kit treated SF samples revealed clearer isoforms and higher intensities for the less abundant proteins of haptoglobin, apolipoprotein A-IV, prostaglandin-D synthase, alpha-1B-glycoprotein, and alpha-2-HS-glycoprotein on 2-DE gels. As a result, it is recommended that SF samples should be treated with protein clean up products such as 2-D Clean-Up Kit first before conducting proteomic research in searching for the relevant biomarkers associated with knee osteoarthritis.

Dataset Information

Proteomic profiling of aging in glomeruli of mice by using two-dimensional differential gel electrophoresis.

Publications

Proteomic profiling of aging in glomeruli of mice by using two-dimensional differential gel electrophoresis.

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