The broad goal of this competing renewal focuses on studying carbohydrate:protein, as well as protein:protein non-covalent interactions. In particular, we propose to investigate the binding and interaction of glycosaminoglycans (GAG) with chemokine ligands of the N-terminal G-protein coupled receptors (GPCR), CCR2 and CCR5, as well as exploring possible ternary complexes of all three using Q-TOF/Ion Mobility Mass Spectrometry. Binding of the GAG with chemokine will be explored via incubation of various GAG libraries with the chemokines of CCR2 and CCR5. Specific binders will be identified using our GAG compositional analysis. Chemokines will be expressed and purified in house. Collisional cross section measurements will be made for any of the non-covalent complexes identified, thus providing information about conformation change upon binding. Additional Surface Plasmon resonance and MS kinetic binding studies along with thermal calorimetry will provide important information about the binding strength. The intent is that a detailed understanding of these interactions will allow for effective targeting of the chemokine system for therapeutic benefit. The major areas proposed are interwoven in such a way that methods development, biotechnology and application to biologically relevant systems are the cohesive elements that bind the proposed research together. PUBLIC HEALTH RELEVANCE: This proposal involves the analysis of non-covalent complexes and interactions of chemokine proteins with sulfated carbohydrates, as well as possible ternary interactions with the N-terminal G-protein coupled receptors. Ion Mobility mass Spectrometry will be used to measure these interactions and to obtain collisional cross section measurements of the binding partners.