Using the Merrifield automated solid phase approach to peptide synthesis, Core A will synthesize at the request of each PI of this Program Project, a total of approximately 30 peptides per year. Using the latest techniques for peptide/protein isolation, characterization and analysis, we propose to provide relatively large quantities (25 to 100 mg) of purified, fully characterized peptides. Preparative peptide purification will be achieved by reversed phase high performance chromatography (RP-HPLC) in two solvent systems (Triethylammonium phosphate and O.l% TFA or O.5% acetic acid for desalting). Determination of purity will be done by RP-HPLC, ion exchange chromatography on FPLC and capillary zone electrophoresis, characterization will use amino acid analysis, mass spectroscopy and Edman degradation if necessary. The peptides synthesized under the aegis of this core will support all six projects and Core C. Peptides will be used to carry out functional studies, to raise specific polyclonal antibodies, and, ultimately to support structural hypotheses. Serious attempts will be made to synthesize phosphopeptides for Project 2. Peptides for the raising of antibodies will be carefully designed in such a way that the same peptide can be unequivocally coupled to a protein carrier through a unique bond to raise antibodies and used for radioimmunoassays, and other antibody based assays. With the consent of the concerned Pi, excess reagents (peptides) will be distributed free of charge to any NIH sponsored researcher that would provide adequate justification for their need. This core will take advantage of the experience of the Pi and his fully functional laboratory for peptide synthesis and characterization. Ample laboratory space equipped with hoods and other small equipment is available. Major pieces of equipment already available include a Jeol FAB Mass spectrometer Model HX-l 10, a P/ACE model 2000 capillary zone electrophoresis, a Perkin Elmer amino acid analyzer, an HF line, a micro balance and two lyophilizers. In this core we request 50% support for the purchase of an ABI automated peptide synthesizer Model 43OA-03, an analytical HPLC from Hewlett Packard and a Prep LC 4000 Preparative Chromatography System from Waters Associates. The Salk Institute recognizes the need for such a Facility and pledges the other 50% of the cost of this equipment. Core A will be under the overall responsibility of Dr. Rivier (4% effort) who will chair the users'committee responsible for setting priorities. It is expected that Core A will be able to operate on a first come, first serve basis. In cases where prioritization will be needed, the decision reached by the majority of the users' committee (excluding the chair) will be implemented. Day to day operation of Core A will be under the supervision of Dr. C. Hoeger (20% effort) who will also work on the synthesis of the phosphopeptides. Synthesis and purification of peptides will be carded out by a Research Assistant 11 (75% effort). Support is also requested for amino acid analysis (10% effort) and mass spectroscopic analysis of the synthetic peptides (5% effort).
