Dataset Information

Two-dimensional phos-tag zymograms for tracing phosphoproteins by activity in-gel staining.

ABSTRACT: Protein phosphorylation is one of the most common post-translational modifications regulating many cellular processes. The phos-tag technology was combined with two-dimensional zymograms, which consisted of non-reducing IEF PAGE or NEPHGE in the first dimension and high resolution clear native electrophoresis (hrCNE) in the second dimension. The combination of these electrophoresis methods was mild enough to accomplish in-gel activity staining for Fe(III)-reductases by NADH/Fe(III)-citrate/ferrozine, 3,3'-Diaminobenzidine/H2O2 or TMB/H2O2 in the second dimension. The phos-tag zymograms can be used to investigate phosphorylation-dependent changes in enzyme activity. Phos-tag zymograms can be combined with further downstream analysis like mass spectrometry. Non-reducing IEF will resolve proteins with a pI of 3-10, whereas non-reducing NEPHGE finds application for alkaline proteins with a pI higher than eight. Advantages and disadvantages of these new methods will be discussed in detail.

SUBMITTER: Meisrimler CN

PROVIDER: S-EPMC4396385 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Two-dimensional phos-tag zymograms for tracing phosphoproteins by activity in-gel staining.

Meisrimler Claudia-Nicole CN Schwendke Alexandra A Lüthje Sabine S

Frontiers in plant science 20150414

Protein phosphorylation is one of the most common post-translational modifications regulating many cellular processes. The phos-tag technology was combined with two-dimensional zymograms, which consisted of non-reducing IEF PAGE or NEPHGE in the first dimension and high resolution clear native electrophoresis (hrCNE) in the second dimension. The combination of these electrophoresis methods was mild enough to accomplish in-gel activity staining for Fe(III)-reductases by NADH/Fe(III)-citrate/ferro ...[more]

PMID: 25926840

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Project description:BACKGROUND:The small intestine is an important human organ that plays a central role in many physiological functions including digestion, absorption, secretion and defense. Duodenal pathologies include, for instance, the ulcer associated to Helicobacter Pylori infection, adenoma and, in genetically predisposed individuals, celiac disease. Alterations in the bowel reduce its capability to absorb nutrients, minerals and fat-soluble vitamins. Anemia and osteopenia or osteoporosis may develop as a consequence of vitamins malabsorption. Adenoma is a benign tumor that has the potential to become cancerous. Adult celiac disease patients present an overall risk of cancer that is almost twice than that found in the general population. These disease processes are not completely known.To date, a two dimensional (2D) reference map of proteins expressed in human duodenal tissue is not yet available: the aim of our study was to characterize the 2D protein map, and to identify proteins of duodenal mucosa of adult individuals without duodenal illness, to create a protein database. This approach, may be useful for comparing similar protein samples in different laboratories and for the molecular characterization of intestinal pathologies without recurring to the use of surgical material. RESULTS:The enrolled population comprised five selected samples (3 males and 2 females, aged 19 to 42), taken from 20 adult subjects, on their first visit at the gastroenterology unit for a suspected celiac disease, who did not turn to be affected by any duodenal pathology after gastrointestinal and histological evaluations. Proteins extracted from the five duodenal mucosal specimens were singly separated by 2D gel electrophoresis. After image analysis of each 2D gel, 179 protein spots, representing 145 unique proteins, from 218 spots tested, were successfully identified by MALDI-TOF ms analysis. Normalized volumes, for each protein, have been reported for every gel. Proteins have been grouped according to their biological/metabolic functions. CONCLUSION:This study represents to date the first detailed and reproducible 2D protein map of human duodenum. Spots identifications, reported in a database, will be helpful to identify the variability in protein expression levels, in isoforms expression, or in post-translational modifications associated to pathology or to a therapy.

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

Two-dimensional phos-tag zymograms for tracing phosphoproteins by activity in-gel staining.

Publications

Two-dimensional phos-tag zymograms for tracing phosphoproteins by activity in-gel staining.

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