# A Novel high resolution MS platform for high-throughput screening of G protein-coupled receptors

> **NIH NIH R01** · BATTELLE PACIFIC NORTHWEST LABORATORIES · 2024 · $340,162

## Abstract

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.

## Key facts

- **NIH application ID:** 10854899
- **Project number:** 5R01GM149650-02
- **Recipient organization:** BATTELLE PACIFIC NORTHWEST LABORATORIES
- **Principal Investigator:** Jon Jacobs
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $340,162
- **Award type:** 5
- **Project period:** 2023-06-15 → 2027-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10854899

## Citation

> US National Institutes of Health, RePORTER application 10854899, A Novel high resolution MS platform for high-throughput screening of G protein-coupled receptors (5R01GM149650-02). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10854899. Licensed CC0.

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