The four main objectives of the Pepfidomics, RIA and Proteomics (PRP) Core are to: 1) train CURE: DDRCC investigators in the use of peptide, radioimmunoassay and proteomic techniques for their own research activities;2) perform services for CURE: DDRCC investigators for studies requiring expensive equipment or requiring too much skill to be performed in individual laboratories (i.e. mass spectral analysis, microsequence analysis, amino acid analysis, etc.);3) provide instrumentation for studies requiring expensive equipment and simple protocols where CURE: DDRCC investigators and staff can be trained to perform experiments (i.e. radioimmunoassay of large numbers of samples, HPLC studies using the same method repeatedly);4) provide unique reagents necessary for CURE: DDRCC investigators to conduct their own research (i.e. radiolabels, synthetic peptides, fluorescent labeled synthetic peptides). Many physiological, pharmacological, and biochemical studies performed at CURE: DDRCC utilize peptide reagents, including biologically active agonists and antagonists, cell permeable mimics of intracellular proteins that interfere with signal transduction steps, enzyme substrates, etc. Proper care and evaluation of these reagents are necessary for valid results. CURE: DDRCC investigators are trained in how to make, store and evaluate stock solutions, how to dilute peptides properly, and how to evaluate the loss or modification of peptides under experimental conditions. The PRP will also provide services and equipment for CURE: DDRCC investigators to acquire state of the art characterization of peptides. A wealth of antibodies has been produced by the Antibody Core during the past funding period, and these continue to be an integral component of several studies performed at CURE: DDRCC. These antibodies will be tested and distributed bythe PRP Core. The personnel of the Core will continue to enable investigators to perform radioimmunoassay and ELISA techniques. The Shared Proteomics Facility is a new addition to the CURE: DDRCC. Purification and characterization of proteins by high performance capillary electrophoresis, high performance liquid chromatography, microsequence analysis, and mass spectral analysis require instrumentation far too costly for individual laboratories and expertise to perform complicated techniques properly and efficiently. Many of the chromatography techniques used for years by Dr. Reeve and his staff (1) are directly applicable to proteomics. This is especially true for low abundance proteins of a cell because 2D gels are limited in volume of sample that can be applied. Liquid chromatography also offers the ability to perform 3D or 4D separations so proteins can be highly purified, resulting in cleaner subsequent mass spectral patterns. The PRP will train CURE: DDRCC investigators in proteomic techniques, perform liquid chromatography, mass spectral analysis and informatics necessary to characterize the proteomes under investigation. The Proteomic facilities include a variety of high performance mass spectrometers capable of providing detailed information on the structure of biomolecules, such as time-of-flight, magnetic sector, quadrupole, triple quadrupole, quadrupole ion trap and quadrupole-time-of-flight (QTOF) tandem mass spectrometers (i.e., MS/MS). These instruments are equipped with conventional electron impact (EI), chemical (CI), atmospheric pressure chemical (APCI) and fast atom bombardment (FAB) ion sources, in addition to electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) sources for the sensitive analysis of peptides and proteins. On-line separation methods such as capillary HPLC are interfaced to ESI-MS systems to characterize low abundance level peptide and protein samples. The Resource also has access to supporting systems for proteomics research, including gel electrophoresis, automated protein digesters, and a robotic MALDI sample spotter. A computer server network used for high speed protein sequence database searching is dedicated for mass spectrometry-based protein identification.
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