Core C will provide essential functions for Projects 1-5 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. Each year, using Merrifield's automated SPPS, Core C will synthesize 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 posttranslational modifications using highly resolving mass spectrometric methods. 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 and posttranslational modifications 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.
This Core fulfills important support functions for all of the projects of this Program by providing highly purified peptides and by characterizing ligands and binding proteins of the CRF/Urocortin system. This system is the major modulator of the mammalian stress response. In addition, it plays an important role in diseases such as diabetes and those of the cardiovascular system.
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