Chemical proteomics strategies for quantifying inflammatory protein networks and modifications - The overall vision of the research program is to investigate inflammatory signaling pathways regulated by pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and NOD-like receptors (NLRs), at the molecular level using innovative mass spectrometry-based chemical proteomics technology. TLRs, NLRs, and other PRRs play central roles in several diseases, including infectious, inflammatory, and cancer. While biomedical research over the past decade has significantly advanced our understanding of the physiological function of several TLRs and NLRs, the precise mechanisms by which diverse PRRs are activated by pathogen- and danger-associated molecular patterns, as well as the biochemical processes and molecular interactions involved in activating downstream signaling pathways, remain poorly understood. Moreover, the physiological functions of many PRRs are yet to be defined. New quantitative proteomics methods are urgently needed to dissect the full spectrum of NLR and TLR host-defense signaling pathways. A systems-level understanding of interactome, and post-translational modification (PTM) networks requires cutting-edge proteomics methods. The major challenge at this moment is the reliable identification and quantification of interactome and PTMs in large-scale studies. Interactome networks are complex and require specific pull-down methods or system-level experiments for unambiguous identification of protein complexes and their modifications. Efficient methods to study transient and weak interactions should be developed to preserve the integrity of protein complexes after cell lysis. PTMs are present in low abundance, and their diverse structures require new methods for confident identification in large-scale samples. Chemistry-based approaches combined with mass spectrometry (MS) have become popular due to their ability to pinpoint interactome and enrich low abundance modifications; furthermore, different features can be incorporated into chemical structures, allowing for confident identification by mass spectrometry. In this regard, gas-phase selective cleavage of chemical bonds in a mass spectrometer has become useful because modified peptides can be traced by a signature mass generated in the mass spectra. This signature mass enables efficient analysis of large-scale samples. This research plan outlines comprehensive chemical strategies designed to quantify system-level protein-to-protein interactions and develop cutting-edge methods to profile post-translational modifications of proteins. These methods will be applied to decipher the host-defense interactome and PTMs of several TLRs and NLRs upon exposure to external and environmental agents. The outcomes of this research will contribute to the design of novel drugs to treat aberrant inflammations caused by excessive activation of TLRs and NLRs and will enable comprehensive study of several biological systems.
