Impact of quenching failure of Cy dyes in differential gel electrophoresis.

BACKGROUND: Differential gel electrophoresis (DIGE) is a technology widely used for protein expression analysis. It is based on labelling with fluorescent Cy dyes. In comparative fluorescence gel electrophoresis experiments, however, unspecific labelling using N-hydroxy-succinimide-ester-based labelling protocols was recently detected. Cross-talk was observed due to failure of the quenching process. Here, the impact of this effect for DIGE experiments was investigated. METHODOLOGY/PRINCIPAL FINDINGS: Experiments to test quenching efficiency were performed in replicate using Escherichia coli lysate. Parameters such as the amount of dye and quencher were varied. Labelling and quenching were reversed in one experiment. Differences in protein spot volumes due to limited quenching were determined. For some spots twice the volume was detected underscoring the importance of proper control of silencing of active dye. CONCLUSIONS/SIGNIFICANCE: It could be demonstrated that uncontrolled labelling increased protein spot volume, even doubling it in some cases. Moreover, proteins responded differently to the protocol. Such unpredictable and unspecific processes are not acceptable in protein regulation studies so that it is necessary to validate the correct amount of quencher for individual samples before the DIGE experiment is performed. Increase of the concentration of lysine, which is used as quencher, from 10 mM to 2500 mM, was sufficient to silence the dye. Alternatively, active dye molecules can be removed by filtration.

False labelling due to quenching failure of N-hydroxy-succinimide-ester-coupled dyes.

In comparative fluorescence gel electrophoresis experiments, cross-talk was detected. It was traced back to a failure in the quenching process in typical labelling protocols. Despite a huge excess of potential reaction sites for the N-hydroxy-succinimide-ester-coupled dye, sufficient active dye molecules were available after the quenching step to label protein molecules un-specifically. It could be shown that only a 100-fold increase in the amount of quencher will silence residual dye to such an extent that no artificial signals are detected.

Homologous housekeeping proteins in Nocardia--the NoDaMS proteomic database.

Nocardiosis is on the rise but hard to diagnose and the application of advanced subtyping technologies is called for. While the genomic sequence for the most virulent strain, Nocardia farcinica is available, proteome data are essentially non-existent. Nevertheless, they are necessary for functional studies on virulence and disease prevention. Here, comparative gel electrophoresis (PAGE)-based analyses of the five Nocardia strains SD1828, N. africana SD910, SD 925, N. sp. 1086, and N. asteroides N317 are discussed. The two-dimensional gel images of all strains are similar and dominated by housekeeping proteins such as chaperones and metabolic enzymes. The sequences of many proteins are highly homologous among strains and in some cases Mycobacterium sequences are closer matches to the unknown than those of N. farcinica. All mass spectrometry data are made available in the NoDaMS database at URL http://ifg.uni-muenster.de/ (Proteomics-Projects-NoDaMS) for re-evaluation with fresh sequencing information. Assignments, homology analyses, and peptide matches are presented. This data review comprises the first comprehensive summary of proteomic data of Nocardia.

Both estrogen receptor subtypes, alpha and beta, attenuate cardiovascular remodeling in aldosterone salt-treated rats.

Experimental and population-based studies indicate that female gender and estrogens protect the cardiovascular system against aldosterone-induced injury. Understanding the function of estrogens in heart disease requires more precise information on the role of both estrogen receptor (ER) subtypes, ERalpha and ERbeta. Therefore, we determined whether selective activation of ERalpha or of ERbeta would confer redundant, specific, or opposing effects on cardiovascular remodeling in aldosterone salt-treated rats. The ERalpha agonist 16alpha-LE2, the ERbeta agonist 8beta-VE2, and the nonselective estrogen receptor agonist 17beta-estradiol lowered elevated blood pressure, cardiac mass, and cardiac myocyte cross-sectional areas, as well as increased perivascular collagen accumulation and vascular osteopontin expression in ovariectomized rats receiving chronic aldosterone infusion plus a high-salt diet for 8 weeks. Uterus atrophy was prevented by 16alpha-LE2 and 17beta-estradiol but not by 8beta-VE2. Cardiac proteome analyses by 2D gel electrophoresis, mass spectrometry, and peptide sequencing identified specific subsets of proteins involved in cardiac contractility, energy metabolism, cellular stress response and extracellular matrix formation that were regulated in opposite directions by aldosterone salt treatment and by different estrogen receptor agonists. We conclude that activation of either ERalpha or ERbeta protects the cardiovascular system against the detrimental effects of aldosterone salt treatment and confers redundant, as well as specific, effects on cardiac protein expression. Nonfeminizing ERbeta agonists such as 8beta-VE2 have a therapeutic potential in the treatment of hypertensive heart disease.