Phosphoproteomic analysis of NADPH oxidase activation
Type of Award: Catalyst
Award Period: July 2010 - June 2012
Amount Awarded: $ 200,000.00
PI(s): Richard Ye, MD, PhD, UIC; Neil Kelleher, PhD, NU;
Abstract: Protein phosphorylation is a major form of post-translational modification known to be important for numerous physiological functions in eukaryotic cells. Top-down mass spectrometry (MS), which analyzes intact proteins, offers several advantages over the traditional bottom-up MS in the characterization of protein phosphorylation. In this application, we propose to extend the top-down MS-based approach from analyzing individual proteins to characterizing multiple proteins for their phosphorylation states within a defined biological system (a "microproteome"). The phagocyte NADPH oxidase is ideally suited for this type of analysis and model development because (1) the essential components of the oxidase complex and their functions have been defined, and (2) the signaling molecules involved in the oxidase assembly and activation have been identified, but their temporal and spatial regulation remains largely uncharacterized. Combining the respective expertise of the two principal investigators in phagocyte biology and MS-based proteomics research, this applications aims to establish novel methodologies for precise analysis of complex biological systems such as those present in phagocytes, cancer cells and stem cells. In Aim 1, we will characterize agonist-dependent phosphorylation of p47phox at multiple sites and develop a model for MS-based analysis of proteins that are extensively and differentially phosphorylated. In Aim 2, we will extend the top-down MS approach from examining individual signaling molecules to simultaneous analysis of multiple kinases within a defined system such as the phagocyte NADPH oxidase. This part of the study takes advantage of autophosphorylation of protein kinases upon activation. Since these phosphorylation events occur at defined sites within the kinase proteins, changes in molecular weight resulting from autophosphorylation at single or multiple sites may be determined rapidly and precisely using top-down MS. A successful completion of the proposed study will not only improve our understanding of the regulatory mechanisms for NADPH oxidase activation, but also obtain much needed information for broad applications of top-down MS-based "precision proteomics" in future studies of complex biological systems.