One of the major goals of the MCW Proteomics Center is to develop new strategies and methods for the separation and isolation of different classes of proteins from cellular lysates. These methods will serve as a preliminary step to mass spectrometric characterization of the proteome or sub-proteomes of mammalian cells.

Initially, our component will focus on:

1. Development of ELISA methods to quantify five different proteins known to be involved in angiogenesis:
   1. Vascular Endothelial Growth Factor (VEGF)
   2. Vascular Endothelial Growth Factor Receptor1 (VEGF-R1)
   3. Vascular Endothelial Growth Factor Receptor2 (VEGF-R2)
   4. Osteopontin (OSTP)
   5. Endothelial Nitric-Oxide Synthase (eNOS)
2. Protocols for immunoprecipitation of individual proteins
3. Methods for efficient isolation of all proteins, membrane-bound proteins, or phosphoproteins from cells
4. Development of mass-spectrometric methods to identify, characterize, and quantify proteins from simple and complex protein mixtures. Special emphasis will be given to the analysis of posttranslational modifications of proteins.

Component 2 will provide rat endothelial cells (rVEC) for these studies. Cells will be obtained by rat hind limb dissection from both parental rat strains and consomic animals. Cells will undergo angiogenic stimuli in vivo or in vitro, and will be tested using assays and protocols developed by our component. Protocols and assays will be developed and distributed in collaboration with Pierce Milwaukee, a division of Perbio Sciences AB.