CORE--SOLID PHASE PEPTIDE SYNTHESIS AND HPLC/CZE

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Using the Merrifield automated solid phase approach to peptide synthesis (SPPS), Core D will synthesize, each year, a total of approximately 20 peptides (10 to 50 residues in length; linear, cyclic, phosphorylated). Using the latest techniques for the preparative (large scale: mg to gram quantities) purification of peptides/proteins, we propose to provide relatively large quantities (10 to 100 mg) of purified peptides/proteins to support investigations carried out in most Projects. Using the latest analytical techniques we will ascertain the purity and identity of all synthetic peptides and proteins including that of peptides to be used clinically. Determination of purity will be done by reverse phase-HPLC, ion exchange chromatography on FPLC and capillary zone electrophoresis (CZE). Characterization will use amino acid analysis (AAA), mass spectroscopy (LSIMS) and Edman degradation (Core C) when necessary. The peptides synthesized under the aegis of this core will support most projects while Core C will use AAA and CZE. Peptides will be used to carry out functional and structural studies as well as to raise specific polyclonal antibodies (See Projects I, II, IV, V). Finally, we intend to use some of the resources of this core to develop new analytical techniques and explore new applications. This core will take advantage of the PI's experience (7% effort) and of that of his technical assistants as well as of his fully functional laboratory for SPPS and characterization. Laboratory space equipped with hoods and other small equipment is available. Major instruments available include a LSI Mass Spectrometer Model HX-110 and a 60 MHz NMR from JEOL, a Beckman P/ACE model 2050 CZE, a Perkin Elmer AAA, four peptide synthesizers from Beckman, analytical and preparative HPLCs and lyophilizers. PI will chair the users' committee responsible for setting priorities. It is expected that Core D will be able to operate on a first come, first served basis. In cases where priority will have to be determined, the decision reached by the majority of the users' committee (excluding the chair) will be implemented. Synthesis and purification of peptides will be carried out by G-C. Jiang (100 % effort). Quality control and method development using HPLC, FPLC and CZE will be carried out by C. Miller (30 % effort). AAA (10 % effort) and MS analysis (15 % effort) will be carried out by D. Pantoja under the supervision of A. Craig (8 % effort). With the consent of the concerned PIs, excess peptides will be distributed free of charge to any NIH sponsored researcher as we have done in the past.