Core E will provide state-of-the-art protein analysis services for SCCOR projects as a high quality, centralized resource to provide consistency and reproducibility in sample preparation, data analysis, and cost savings by eliminating duplications in equipment and reagents. Core E is based in the Biomolecular Resource Facility (BRF), an established Institutional Core Laboratory of the University of Texas Medical Branch that is partially funded by the NHLBI Proteomics Center contract mechanism. The BRF occupies 6,200 ft2 distributed within twelve laboratories and seven offices located centrally in the campus at UTMB. The current scientific staff includes 17 (6 Ph.D., 3 M.S., and 6 B.S.) individuals. Specifically, the Proteomics Core will provide established methodologies for sample pre-separation fractionation, 2-dimensional gel electrophoresis (2DE), differential protein staining, gel imaging and analysis, peptide labeling with stable isotopes (iTRAQ) and quantitative mass spectrometry, protein identification by peptide mass fingerprinting via matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS), liquid chromatography tandem mass spectrometry (LC/MS/MS), and Luminex multiplex assays for determination of cytokine expression patterns. The specific objectives of the Proteomics Core are: 1. Provide the infrastructure necessary for consistent sample pre-separation fractionation (Project 1);2. Perform differential protein expression analysis by 2DE and identification of proteins from vascular smooth muscle cells and aortic explant cultures (Projects 1 and 3);3. Perform differential iTRAQ labeling, LC/MS/MS protein identification of complex protein samples (Projects 1 and 3);and 3. Perform immunoassays of human and mouse chemokines/cytokines in aortic explant cultures from human and mouse samples (Projects 3 and 4). To ensure data quality, validation activities using standard proteins and/ or peptides have been established. Validation of data generated by image analysis with Progenesis software is accomplished utilizing appropriate statistical analyses including Student's t-test for hypothesis and significance testing, Multiple Hypothesis testing corrections, Hierarchical Clustering of control vs. treated, and Analysis of Variance (ANOVA) for time course/dosage dependence of expression. For MS, data quality is ensured through the use of appropriate internal and external standards, while MS instruments are routinely calibrated with external standards. For the Bioplex cytokine measurements, recombinant standards are run with each plate and sample-to-sample variation determined.
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