The goal of the proposed research is the structural analysis of proteins isolated by chromatographic methods from natural sources, by blotting from one- and two-dimensional gels, and by expression of recombinant cDNA's in bacterial, yeast, or insect cells. This will be accomplished by new instrumentation consisting of a Milligen Model 6625 protein sequencer and a Hewlett-Packard Model 1090M high performance liquid chromatograph (HPLC). The instruments will be located in and operated by the Protein Chemistry Laboratory at Cold Spring Harbor Laboratory. The projects described by the user group represent large projects that cannot be accommodated by the protein sequencing Core facility. These projects include 1) systematic purification and structural analysis of proteins involved in human DNA replication; 2) the purification and analysis of proteins responsible for pre-mRNA splicing; 3) characterization of protein tyrosine phosphatases; 4) structure-function studies of protein kinases; 5) analysis of phosphorylation sites on nuclear oncoproteins and other proteins related to cell growth and differentiation; and 6) the identification and characterization of signal transduction and cell cycle proteins detected on high resolution, two-dimensional gels. All of these projects involve multiple proteins, and they require protein sequencing of peptides derived from these proteins and purified by HPLC. In addition, all the projects include the analysis of phosphorylation sites that are found on the proteins of interest. The protein sequencer requested is shown to be useful at sequencing phosphorylation sites, including phospho-tyrosine. The HPLC requested is equipped to handle narrow bore columns and has a diode-array detector that can be optimized for sensitivity or resolution in the preparation of peptide fragments for sequencing. The requested instrumentation will permit the analysis of proteins that are fundamental to basic molecular mechanisms within the cell.
