The proteomics Core D of the UAB Skin Disease Research Center provides support to pilot/feasibility awardees, SDRC-associated investigators and other skin disease researchers. This support consists of consultations imvolving two experienced senior faculty with expertise in 2D-gel proteomics and protein mass spectrometry, hands-on support by a junior faculty member skilled in several proteomics/mass spectrometry methods used and applied to skin disease research, training in the techniques offered by the Core, access to the instrumentation, and seminars by outside experts in proteomic and mass spectrometry. The specific services available in the Core include (1) development of experimental design and statistical considerations for experiments that include proteomics and mass spectrometry components;(2) 2D-gel separation of proteins from tissues, cells, subcellular fractions and specific body fluids (plasma/serum, urine, sweat, tears, tissue interstitial and blister fluids) - this involves isoelectric focusing/SDS-PAGE or clear native gel electrophoresis and makes use of Cy2, Cy3 and Cy5 fluorescent dyes and Sypro Ruby red post-staining, imaging and image analysis using DeCyder software, robotic gel spot picking, chemical processing and protease digestion, and peptide mass fingerprinting;(3) 1D-SDS-PAGE gel separation of membrane-associated proteins combined with nanoLC-ESI-tandem mass spectrometry;(4) isotope-labeling methods (iTRAQ reagents) to carry out quantitative analysis of the effects of treatments and time on members of the tissue, cell and fluid proteomes; and (5) development of specific multiple reaction ion monitoring (MRM) mass spectrometry methods to quantify peptides that allow quantitative analysis of individual, specified proteins. In addition, the LC-MRM-MS methods can be applied to the analysis of the reagents used in skin disease research, for example, green tea polyphenols and grape seed extract, as well as their and drug metabolites in the blood and other fluids. A special emphasis is made in maintaining quality control of the methods. A Laboratory Information Management System will be used to oversee the operations of the Core. Investigators will be trained individually in the use of the Core techniques as well as at workshops and a Symposium held in conjunction with the other NIH-funded Centers
The science of skin disease research will be substantially improved by providing this resource in proteomics. It brings to the investigators well developed, state-of-the-art methods to better understand the events that comprise the disease process. It will help define strategies that should lead to improvements in the prevention and treatment of skin diseases.
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