Comparative Two-Dimensional Fluorescence Gel Electrophoresis.

Two-dimensional comparative fluorescence gel electrophoresis (CoFGE) uses an internal standard to increase the reproducibility of coordinate assignment for protein spots visualized on 2D polyacrylamide gels. This is particularly important for samples that need to be compared without the availability of replicates and thus cannot be studied using differential gel electrophoresis (DIGE). CoFGE corrects for gel-to-gel variability by co-running with the sample proteome a standardized marker grid of 80-100 nodes, which is formed by a set of purified proteins. Differentiating of reference and analyte is possible by the use of two fluorescent dyes. Variations in the y-dimension (molecular weight) are corrected by the marker grid. For the optional control of the x-dimension (pI), azo-dyes can be used. Experiments are possible in both vertical and horizontal (h) electrophoresis devices, but hCoFGE is much easier to perform. The CoFGE experimental principle can additionally be used for protein quantification. For data analysis, commercial software has been adapted.

Redox proteomics reveals an interdependence of redox modification and location of adhesome proteins in NGF-treated PC12 cells.

Proteomics studies have revealed that adhesomes are assembled from a plethora of proteins at integrin-mediated cellular contact sites with the extracellular matrix. By combining dimedone-trapping of sulfenylated proteins with the purification of the adhesome complex, we extended previous proteomics approaches on adhesomes to a redox proteomic analysis. This added a new aspect of adhesome complexity as individual adhesome proteins change their redox state in response to environmental signals. As model system, rat pheochromocytoma PC12 cells were studied in contact with type IV collagen and in response to nerve growth factor (NGF). NGF stimulates the endogenous production of reactive oxygen species (ROS) and the formation of neurite-like cell protrusions, which are anchored to the substratum via adhesomes. Dimedone detects the reversible oxidation of cysteine thiol groups into sulfenic acid groups which was used in proteomic analysis of adhesome proteins revealing that sulfenylation and location of proteins mutually influence each other. For some proteins, identified by the redox proteomics approach, among them Nck-associated protein-1 (Nap-1), proximity ligation analysis and co-immunoprecipitation assays proved that protein sulfenylation sites colocalize with adhesomes of protrusions. In conclusion, the suprastructural composition and function of adhesomes is redox-regulated by ROS. Of interest in this respect, isoform-selective pharmacological inhibition of NADPH-oxidases (Noxs) reduced the adhesomal location of the collagen-binding α1ß1 integrin and the length of the outgrowing neurites, indicative of a role of Nox isoforms in the redox-regulation of adhesomes. Thus, our novel redox proteomics approach not only revealed redox-modifications and the potential redox-regulation of adhesomes and their constituents but it may also provide a tool to analyze the ROS-stimulated neurite repair of peripheral neurons.

Identification of Ocular Autoantigens Associated With Juvenile Idiopathic Arthritis-Associated Uveitis.

The purpose of the current study was to analyze the binding patterns of serum autoantibodies from juvenile idiopathic arthritis (JIA) and JIA-associated uveitis (JIAU) patients to proteomes from different ocular tissues and to identify potential ocular autoantigens in JIAU. Proteomes from porcine iris, ciliary body, or retina tissue were isolated, separated using 2D-gel electrophoresis, and transferred to a blotting membrane. The binding pattern of serum antibodies from JIA or JIAU patients or healthy controls to ocular proteins was visualized by using anti-human IgG secondary antibodies and chemiluminescence reaction. Selected protein spots were excised from silver-stained 2D gels and subjected to mass spectrometry. Serum antibodies binding to ocular proteins were detected in all patient groups and healthy controls. Irrespective of the patient groups, serum antibodies bound to 49 different protein spots of the retina proteome, to 53 of the ciliary body proteome, and to 44 of the iris proteome. The relative binding frequency of sera to these iris protein spots was significantly higher in JIAU than in JIA patients or healthy controls. Particularly in JIAU patients, cluster analyses indicated a broad range of serum antibodies directed against ocular antigens, mostly in the iris proteome. Iris proteins frequently bound by serum antibodies in all groups were identified as tubulin beta chain, vimentin, ATP synthase subunit beta, actin, and L-lactate dehydrogenase B chain. Iris proteins exclusively bound by JIAU serum antibodies were heat shock cognate 71 kDa protein and keratin. Although serum autoantibody binding to ocular antigens was not disease-specific, a significant diversity of autoantibodies against a broad range of antigens, particularly from the iris tissue, was detected in JIAU patients. As the iris is a major site of inflammation in JIAU, the present data give further evidence that autoantibodies may be involved in JIAU immunopathology.

Transcriptomic and proteomic analysis of iris tissue and aqueous humor in juvenile idiopathic arthritis-associated uveitis.

Gene and protein expression profiles of iris biopsies, aqueous humor (AqH), and sera in patients with juvenile idiopathic arthritis-associated uveitis (JIAU) in comparison to control patients with primary open-angle glaucoma (POAG) and HLA-B27-positive acute anterior uveitis (AAU) were investigated. Via RNA Sequencing (RNA-Seq) and mass spectrometry-based protein expression analyses 136 genes and 56 proteins could be identified as being significantly differentially expressed (DE) between the JIAU and POAG group. Gene expression of different immunoglobulin (Ig) components as well as of the B cell-associated factors ID3, ID1, and EBF1 was significantly upregulated in the JIAU group as compared to POAG patients. qRT-PCR analysis showed a significantly higher gene expression of the B cell-related genes CD19, CD20, CD27, CD138, and MZB1 in the JIAU group. At the protein level, a significantly higher expression of Ig components in JIAU than in POAG was confirmed. The B cell-associated protein MZB1 showed a higher expression in JIAU patients than in POAG which was confirmed by western blot analysis. Using bead-based immunoassay analysis we were able to detect a significantly higher concentration of the B cell-activating and survival factors BAFF, APRIL, and IL-6 in the AqH of JIAU and AAU patients than in POAG patients. The intraocularly upregulated B cell-specific genes and proteins in iris tissue suggest that B cells participate in the immunopathology of JIAU. The intracameral environment in JIAU may facilitate local effector and survival functions of B cells, leading to disease course typical for anterior uveitis.

Comparative Two-Dimensional Fluorescence Gel Electrophoresis.

Two-dimensional comparative fluorescence gel electrophoresis (CoFGE) uses an internal standard to increase the reproducibility of coordinate assignment for protein spots visualized on 2D polyacrylamide gels. This is particularly important for samples, which need to be compared without the availability of replicates and thus cannot be studied using differential gel electrophoresis (DIGE). CoFGE corrects for gel-to-gel variability by co-running with the sample proteome a standardized marker grid of 80-100 nodes, which is formed by a set of purified proteins. Differentiation of reference and analyte is possible by the use of two fluorescent dyes. Variations in the y-dimension (molecular weight) are corrected by the marker grid. For the optional control of the x-dimension (pI), azo dyes can be used. Experiments are possible in both vertical and horizontal (h) electrophoresis devices, but hCoFGE is much easier to perform. For data analysis, commercial software capable of warping can be adapted.

Comparative fluorescence two-dimensional gel electrophoresis using a gel strip sandwich assembly for the simultaneous on-gel generation of a reference protein spot grid.

The comparison of proteins separated on 2DE is difficult due to gel-to-gel variability. Here, a method named comparative fluorescence gel electrophoresis (CoFGE) is presented, which allows the generation of an artificial protein grid in parallel to the separation of an analytical sample on the same gel. Different fluorescent stains are used to distinguish sample and marker on the gel. The technology combines elements of 1DE and 2DE. Special gel combs with V-shaped wells are placed in a stacking gel above the pI strip. Proteins separated on the pI strip are electrophoresed at the same time as marker proteins (commercially available purified protein of different molecular weight) placed in V-wells. In that way, grids providing approximately 100 nodes as landmarks for the determination of protein spot coordinates are generated. Data analysis is possible with commercial 2DE software capable of warping. The method improves comparability of 2DE protein gels, because they are generated in combination with regular in-gel anchor points formed by protein standards. This was shown here for two comparative experiments with three gels each using Escherichia coli lysate. For a set of 47 well-defined samples spots, the deviation of the coordinates was improved from 7% to less than 1% applying warping using the marker grid. Conclusively, as long as the same protein markers, the same size of pI-strips and the same technology are used, gel matching is reproducibly possible. This is an important advancement for projects involving comparison of 2DE-gels produced over several years and in different laboratories.

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.

Identification of poly(ADP-ribose)polymerase-1 and Ku70/Ku80 as transcriptional regulators of S100A9 gene expression.

BACKGROUND: S100 proteins, a multigenic family of non-ubiquitous cytoplasmic Ca2+-binding proteins, have been linked to human pathologies in recent years. Dysregulated expression of S100 proteins, including S100A9, has been reported in the epidermis as a response to stress and in association with neoplastic disorders. Recently, we characterized a regulatory element within the S100A9 promotor, referred to as MRE that drives the S100A9 gene expression in a cell type-specific, activation- and differentiation-dependent manner (Kerkhoff et al. (2002) J. Biol. Chem. 277, 41879-41887). RESULTS: In the present study, we investigated transcription factors that bind to MRE. Using the MRE motif for a pull-down assay, poly(ADP-ribose)polymerase-1 (PARP-1) and the heterodimeric complex Ku70/Ku80 were identified by mass spectrometry and confirmed by chromatin immunoprecipitation. Furthermore, TPA-induced S100A9 gene expression in HaCaT keratinocytes was blocked after the pharmacologic inhibition of PARP-1 with 1,5-isoquinolinediol (DiQ). CONCLUSION: The candidates, poly(ADP-ribose)polymerase-1 (PARP-1) and the heterodimeric complex Ku70/Ku80, are known to participate in inflammatory disorders as well as tumorgenesis. The latter may indicate a possible link between S100 and inflammation-associated cancer.