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  • Pilot Application of iTRAQ to the Retinal Disease Macular Telangiectasia. 22098468

    We used the comparative proteomic technique iTRAQ coupled with offline 2DLC-MS/MS to analyze a rare specimen of the poorly understood, potentially blinding ophthalmic condition Macular Telangiectasia type 2 (MacTel type 2). We refined the technique using an internal standard consisting of pooled samples for each iTRAQ experiment to allow for multiple comparisons between different regions of the retina and different tissue donors. A total of 594 nonredundant proteins were identified in the retina and 168 in the vitreous, of which approximately half were found in significantly different abundance in the various comparisons made. The most prominent differences were found within the glycolytic pathway, where 8 proteins were reduced in the diseased macula compared with peripheral retina of the same eye, and 10 were also reduced in comparison with the macula of a control eye. Furthermore, Müller cell-associated proteins, including GFAP, VIME, and GLNA, were also reduced in the diseased macula, consistent with a link between the glycolytic pathway and Müller cells. These changes were validated by Western blotting and immunohistochemical studies. Proteomic analysis of the vitreous revealed an increase of proteins that were reduced in the retina. This supports proteomic analysis of the more easily available vitreous, which may reveal retina-specific protein changes associated with disease. Furthermore, our study has highlighted changes in the glycolytic pathway as a possible component of MacTel type 2 pathobiology.
    Document Type:
    Reference
    Product Catalog Number:
    MAB302
    Product Catalog Name:
    Anti-Glutamine Synthetase Antibody, clone GS-6

  • Efficient organic monoliths prepared by γ-radiation induced polymerization in the evaluation of histone deacetylase inhibitors by capillary(nano)-high performance liquid ... 21561626

    New monolithic HPLC columns were prepared by γ-radiation-triggered polymerization of hexyl methacrylate and ethylene glycol dimethacrylate monomers in the presence of porogenic solvents. Polymerization was carried out directly within capillary (250-200μm I.D.) and nano (100-75μm I.D.) fused-silica tubes yielding highly efficient columns for cap(nano)-LC applications. The columns were applied in the complete separation of core (H2A, H2B, H3, and H4) and linker (H1) histones under gradient elution with UV and/or electrospray ionization (ESI) ion trap mass spectrometry (MS) detections. Large selectivity towards H1, H2A-1, H2A-2, H2B, H3-1, H3-2 and H4 histones and complete separation were obtained within 8min time windows, using fast gradients and very high linear flow velocities, up to 11mm/s for high throughput applications. The method developed was the basis of a simple and efficient protocol for the evaluation of post-translational modifications (PTMs) of histones from NCI-H460 human non-small-cell lung cancer (NSCLC) and HCT-116 human colorectal carcinoma cells. The study was extended to monitoring the level of histone acetylation after inhibition of Histone DeACetylase (HDAC) enzymes with suberoylanilide hydroxamic acid (SAHA), the first HDAC inhibitor approved by the FDA for cancer therapy. Attractive features of our cap(nano)-LC/MS approach are the short analysis time, the minute amount of sample required to complete the whole procedure and the stability of the polymethacrylate-based columns. A lab-made software package ClustMass was ad hoc developed and used to elaborate deconvoluted mass spectral data (aligning, averaging, clustering) and calculate the potency of HDAC inhibitors, expressed through a Relative half maximal Inhibitory Concentration parameter, namely R_IC(50) and an averaged acetylation degree.Copyright © 2011 Elsevier B.V. All rights reserved.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
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