Core--Analytical and Peptide Synthesis

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This Core C will provide essential functions for the Projects of this Program Project. These include the synthesis of peptides that are required by the Projects for biological and biochemical investigations. In addition, the Core will characterize synthetic, natural or modified peptides and proteins by mass spectrometry and Edman based chemical sequencing. Core C will synthesize and characterize peptides, and develop new HPLC- and CZE-based analytical systems to improve resolution and efficiency. Additionally, this Core will perform circular dichroism experiments as requested. Using the Merrifield automated SPPS, Core C will synthesize, each year, a total of approximately 10-15 peptides (20 to 40 residues in length; linear, cyclic, phosphorylated). Using the latest techniques for the preparative purification of peptides/proteins, we will provide 10 to 100 mg of highly purified peptides to support investigations carried out in most Projects. All peptides will be extensively characterized by the techniques available to the Core, including HPLC, CZE, MS, and Edman degradation. The protein chemical characterization of peptides and proteins involved in neuroendocrine regulation is an essential part of this Program. The Core provides both state-of-the-art instrumentation and highly trained investigators and support personnel to carry out these characterizations. The Core will characterize peptides, proteins and their post-translational modifications. In particular, the disulfide arrangement of soluble forms of CRFRs, both recombinant and naturally occurring, will be determined. In addition, we will confirm the covalent structure of recombinant proteins that the Projects generate for use in biological studies. The processing of urocortins will be established by isolating the peptides from tissues or cell lines and determining their primary structure by mass spectrometry and Edman degradation. The Core will use its proteomics capability to define proteins interacting with the CRF receptor system. Affinity tagged receptors and receptor fragments will be designed and transfected into cells. Complexes will be isolated and their protein constituents will be identified by mass spectrometry.