Bovine dilated cardiomyopathy: Proteomic analysis of an animal model of human dilated cardiomyopathy

Weekes, John; Wheeler, Colin; Yan, Jun X.; Weil, Joachim; Eschenhagen, Thomas; Scholtysik, G; Dünn, Michael J.

doi:10.1002/(sici)1522-2683(19990101)20:4/5<898::aid-elps898>3.0.co;2-b

Cited by 132 publications

(45 citation statements)

References 33 publications

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“…Cells were lysed in 2-DE lysis buffer and the protein concentration was determined by modified Bradford assay (Weekes et al, 1999). In all, 50 mg of protein was loaded onto a 12% SDS polyacrylamide gel and electrophoretically transferred onto a nitrocellulose membrane (Transblot, Biorad).…”

Section: Western Blot Analysismentioning

confidence: 99%

Androgens target prohibitin to regulate proliferation of prostate cancer cells

et al. 2004

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Proteins involved in the growth response of prostate cancer cells to androgen were investigated by comparing the proteomes of LNCaP cells treated with vehicle or androgen. Whole-cell lysates were separated by twodimensional PAGE, and HPLC-MS/MS was used to identify androgen-regulated proteins. Prohibitin, a protein with cell-cycle regulatory activity, was shown to be downregulated by 50% following androgen stimulation. Western blot and reverse transcription-PCR experiments confirmed the result and showed that regulation occurs at the level of transcription. To determine the importance of prohibitin in androgen-stimulated growth, we used transient transfection to overexpress the protein and RNA interference to knock down the protein. Subsequent FACS analysis showed that cells with reduced levels of prohibitin showed a slight but reproducible increase in the percentage of population in cell cycle, while cells with increased prohibitin levels showed a clear reduction in the percentage entering cell cycle, following dihydrotestosterone stimulation, when compared to untransfected controls. Confocal microscopy showed localization of prohibitin in the nucleus as well as the mitochondria of LNCaP cells. It therefore seems that the regulation of prohibitin is a vital part of the cellular growth response to androgen stimulation in LNCaPs and prohibitin may have a nuclear regulatory role in cell-cycle progression.

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Section: Western Blot Analysismentioning

confidence: 99%

Androgens target prohibitin to regulate proliferation of prostate cancer cells

et al. 2004

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“…Bovine dilated cardiomyopathy serves as a model for human dilated cardiomyopathy (DCM). Quantitative alterations in protein concentrations have been determined between healthy and diseased bovine left ventricular tissue using 2-DE [17]. The proteome analysis of Helicobacter pylori and other bacterial pathogens is reviewed [18,19], as well as proteomics and human diseases [20].…”

Section: Investigating Gene Expression: Proteome Analysismentioning

confidence: 99%

Possibilities to improve automation, speed and precision of proteome analysis: A comparison of two-dimensional electrophoresis and alternatives

2001

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Proteome analysis requires fast methods with high separation efficiencies in order to screen the various cell and tissue types for their proteome expression and monitor the effect of environmental conditions and time on this expression. The established two-dimensional gel electrophoresis (2-DE) is by far too slow for a consequential screening. Moreover, it is not precise enough to observe changes in protein concentrations. There are various approaches that promise faster, automated proteome analysis. This article concentrates on capillary (CT isoelectric focusing coupled to mass spectrometry (CIEF-MSn) and preparative IEF followed by size-exclusion chromatography, hyphenated with MS (PIEF-SEC-MS). These two approaches provide a similar separation pattern as the established 2-DE technique and therefore allow for the continued use of data based on this traditional approach. Their performances have been discussed and compared to 2-DE, evaluating 169 recent articles. Data on analysis time, automation, the detection limit, quantitation, peak capacity, mass and pI accuracy, as well as on the required sample amount are compared in a table.

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“…Human heart (left ventricle) proteins were prepared from a tissue sample of unused transplant donor organ and subjected to 2-D PAGE essentially according to Görg et al [6] and Weekes et al [10]. Duplicate analytical loadings of broad-range (3-10NL; 100 g total protein), narrow-range (4-7L, 6-9L; 100 mg total protein) and very narrow-range IPGs (3.5-4.5L, 4-5L, 4.5-5.5L, 5-6L, 5.5-6.7L; 200 g total protein) were used.…”

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confidence: 99%

Zooming-in on the proteome: Very narrow-range immobilised pH gradients reveal more protein species and isoforms

Westbrook¹,

Yan²,

Wait³

et al. 2001

Electrophoresis

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142

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Two-dimensional gel electrophoresis (2-DE) enables separation of complex mixtures of proteins on a single polyacrylamide gel according to isoelectric point, molecular weight, solubility, and relative abundance. For this reason, 2-DE together with mass spectrometry (MS) has become a key technology in proteome analysis. The introduction of immobilised pH gradients (IPGs) for isoelectric focusing of proteins affords improved reproducibility and permits full-scale proteome analyses to be undertaken. Whilst broad-range IPGs are useful for investigating simple proteomes (e.g. Mycoplasma genitalium) it is becoming clear that additional resolving power is needed for separating the more complex proteomes of eukaryotic organisms. The use of narrow-range and very narrow-range IPGs provides the means with which to dissect a complex proteome. We have compared very narrow-range IPGs (3.5-4.5L, 4-5L, 4.5-5.5L, 5-6L, and 5.5-6.7L) with broad- (3-10NL) and narrow-range IPGs (4-7L and 6-9L) for the visualisation of the human heart proteome. The superior ability of very narrow-range IPGs to separate different protein species and isoforms, compared with 3-10NL and 4-7L 2-D gels is demonstrated. The results are supported by MS identifications which further show that reduction of the number of comigrating protein species results in less ambiguous and more reliable database search results.

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Bovine dilated cardiomyopathy: Proteomic analysis of an animal model of human dilated cardiomyopathy

Cited by 132 publications

References 33 publications

Androgens target prohibitin to regulate proliferation of prostate cancer cells

Androgens target prohibitin to regulate proliferation of prostate cancer cells

Possibilities to improve automation, speed and precision of proteome analysis: A comparison of two-dimensional electrophoresis and alternatives

Zooming-in on the proteome: Very narrow-range immobilised pH gradients reveal more protein species and isoforms

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