Summary: Mass spectrometry (MS) has proven invaluable in studying the mechanisms of cellular signaling as MS platforms can directly provide amino acid residue site-specific phosphorylation data compared to traditional antibody-based approaches. However, limitations exist in current MS approaches in generating confident site-specific phosphorylation quantification. This is particularly evident in complex multi-phosphorylated protein motifs, where the detection of isomeric multi-phosphorylated peptides easily overwhelms any prediction scoring approach that is simply based upon the fragmentation spectra. There are many biological examples of hyperphosphorylated regions, where they are associated with receptor/ligand interactions, including G-protein coupled receptors (GPCRs), membrane receptors that are the most common targets for FDA-approved drugs. For accurate site-specific quantification of protein hyperphosphorylation we propose a transdisciplinary approach using ultrahigh resolution Ion Mobility Separation (IMS) integrated with highly accurate and sensitive MS and MS/MS spectra to enable the confident characterization of hyperphosphorylated GPCR ensembles with greatly improved sensitivity, and speed. We will use multi-level Structures for Lossless Ion Manipulations (SLIM) technology (SLIM-Orbitrap platform) to fully characterize phosphorylation of GPCR/antagonist interactions utilizing CXCR3, which plays a central role in inflammatory diseases through its regulation of T cell function as an initial test case. We plan to first integrate ultrahigh resolution IMS with an advanced Orbitrap MS platform for unambiguous decoding of hyperphosphorylated sites, evaluate the SLIM-Orbitrap MS platform for resolving hyperphosphorylated protein regions, and finally, perform comprehensive site-specific phosphoproteomics for GPCRs through screening of activated T cells with dose-responses of chemokine and small-molecule CXCR3 biased agonists.