Development and Applications of Bioorthogonal Chemistry: Administrative Supplement for Equipment ABSTRACT We have a long-standing interest in developing reactivity-based chemical tools to address significant biological problems that are difficult to solve using conventional molecular biology techniques. In our original MIRA application, we plan to continue our studies of orthogonal chemical reactivity at the chemistry-biology interface and pursue the following two related projects. In Project 1, we will construct the FRET-based biosensors of GLP-1R and GCGR using bioorthogonal chemistry techniques. These biosensors will then be employed to probe the conformational dynamics during the ligand-induced receptor activation and signaling in live cells. A new set of fluorescence ?turn-on? reagents will be designed for bioorthogonal labeling of the intracellular loop 3 of GLP-1R and GCGR to allow single-cell intra- and inter-molecular FRET analysis of receptor dynamics in live cells. In Project 2, we will develop genetically encoded chemical crosslinker containing an alkyne group and apply this chemical crosslinker to map the time-dependent GLP-1R and ?-arrestins interactomes by mass spectrometry in response to ligand stimulation. We expect that these studies will validate new bioorthogonal tools for real-time monitoring of protein conformation and protein-protein interactions in live cells. Also, we will gain novel insights into the GLP-1R and GCGR activation dynamics and structural basis of biased signaling that are crucial for the development of targeted therapies for the treatment of diabetes and obesity. This Administrative Supplement requests the acquisition of an Agilent QTOF 6530B LC/MS system, which would allow us to characterize the incorporation of chemical crosslinkers into recombinant proteins with higher mass accuracy and throughput and significantly accelerate the discovery of the GLP-1R and ?-arrestins interactomes by mass spectrometry as described in Project 2.