Core E will provide state-of-the-art protein analysis services for SCCOR projects as a high quality, centralizedresource to provide consistency and reproducibility in sample preparation, data analysis, and cost savings byeliminating 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 bythe NHLBI Proteomics Center contract mechanism. The BRF occupies 6,200 ft2 distributed within twelvelaboratories and seven offices located centrally in the campus at UTMB. The current scientific staff includes 17 (6Ph.D., 3 M.S., and 6 B.S.) individuals. Specifically, the Proteomics Core will provide established methodologiesfor sample pre-separation fractionation, 2-dimensional gel electrophoresis (2DE), differential protein staining, gelimaging and analysis, peptide labeling with stable isotopes (iTRAQ) and quantitative mass spectrometry, proteinidentification by peptide mass fingerprinting via matrix assisted laser desorption ionization time of flight massspectrometry (MALDI TOF MS), liquid chromatography tandem mass spectrometry (LC/MS/MS), and Luminexmultiplex assays for determination of cytokine expression patterns. The specific objectives of the Proteomics Coreare: 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 musclecells and aortic explant cultures (Projects 1 and 3); 3. Perform differential iTRAQ labeling, LC/MS/MS proteinidentification of complex protein samples (Projects 1 and 3); and 3. Perform immunoassays of human and mousechemokines/cytokines in aortic explant cultures from human and mouse samples (Projects 3 and 4). To ensuredata quality, validation activities using standard proteins and/ or peptides have been established. Validation ofdata generated by image analysis with Progenesis software is accomplished utilizing appropriate statisticalanalyses 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/dosagedependence of expression. For MS, data quality is ensured through the use of appropriate internal and externalstandards, while MS instruments are routinely calibrated with external standards. For the Bioplex cytokinemeasurements, recombinant standards are run with each plate and sample-to-sample variation determined.
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