The generation of purified proteins, assessment of protein post-translational modifications (PTMs), protein-protein interactions and evaluation of cellular mechanical properties are integral to the successful completion of the Specific Aims proposed in all three PPG projects. The Protein Chemistry & Biophysics Core will provide services that will support these analyses in all of the PPG Projects.
The Specific Aims of Core D are: 1) Generation of purified recombinant proteins; 2) Analysis of protein PTMs in mechanical stress- challenged cells and tissues; 3) Analysis of protein-protein interactions; 4) Analysis of changes in micromechanical properties of endothelial cells (EC) induced by barrier modifying agents. All three Projects will utilize recombinant proteins for PTM and protein interaction analyses. Proteins generated will be available for each project and for ?in core? use. Additionally, in this aim, proteins from biological samples will be purified to assay PTMs and protein-protein interactions in vivo. To analyze PTMs, we will utilize a mass spectrometry approach coupled to the detection and quantification of specific sites of modification by tandem mass spectrometry. To enhance the analysis of protein-protein interactions (interactome) required in all three Projects, Core D will utilize surface plasmon resonance (SPR) techniques. In vivo measurements of protein- protein interactions will also be validated using immunoprecipitation?mass spectrometry identification approaches. Core D will also utilize atomic force microscopy (AFM) to evaluate structural and elasticity effects in the endothelium to enable investigators to define the relationship between cell mechanics and cytoskeletal reorganization in response to mechanical stretch and the effects of barrier protective agents. Core D leaders have considerable experience with all of the proposed techniques.
The production of purified recombinant proteins will facilitate the identification of novel post-translational modifications and novel protein-protein interactions using mass spectrometry and surface plasmon resonance (SPR) approaches. In addition, atomic force microscopy (AFM) will be used to evaluate how mechanical stress alters the structure and elasticity of EC. These services are best centralized given their inherent difficulty, reliance on specialized and sophisticated equipment and expertise, as well as the requirements of all three projects. Core D will provide these services for all three projects.