Capillary Zone Electrophoresis-Mass Spectrometry of Intact G Protein-Coupled Receptors Enables Proteoform Profiling

Ashley N. Ives; Kevin Jooß; Rafael Donadelli Melani; Ryan T. Fellers; John Janetzko; Neil L. Kelleher

doi:10.1021/acs.analchem.4c06994

Capillary Zone Electrophoresis-Mass Spectrometry of Intact G Protein-Coupled Receptors Enables Proteoform Profiling

Ashley N. Ives, Kevin Jooß, Rafael Donadelli Melani, Ryan T. Fellers, John Janetzko^*, Neil L. Kelleher^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

Abstract

G protein-coupled receptors (GPCRs) are the largest class of integral membrane receptors and responsible for transmitting diverse signals in response to extracellular stimuli. Post-translational modifications serve to dictate the subcellular trafficking and function of a GPCR across space and time. Despite significant interest in mapping the diversity of GPCR modification states (proteoforms), technical challenges have hindered this characterization. While advancements in membrane mimetics and mass spectrometry instrumentation have improved analysis, current workflows require large amounts of homogeneous protein, limiting the study of many GPCRs from mammalian sources. Here, we present capillary zone electrophoresis (CZE) as a separation technique for characterizing proteoforms of both intact and partially digested GPCRs. This method allowed for the characterization of multiple proteoforms of both the β2-adrenergic receptor and metabotropic glutamate receptor 2 using low sample volumes and without buffer optimization. Notably, in the case of smaller phosphorylated analytes, CZE can readily separate positional phosphorylation isomers and provide superior fragmentation coverage to conventional reversed phase-liquid chromatography.

Original language	English (US)
Pages (from-to)	7307-7316
Number of pages	10
Journal	Analytical Chemistry
Volume	97
Issue number	13
DOIs	https://doi.org/10.1021/acs.analchem.4c06994
State	Published - Apr 8 2025

Funding

We thank E. White for assistance with the expression and purification of \u03B22AR. We thank B. Kobilka for valuable discussions of this work. This work was supported by NIH grants T32GM105538 and F31MH129114-01 (to A.N.I.), K99GM147609 (to J.J.) and P41GM108569 (to N.L.K.). J.J. was also supported by a Fellowship Award from the Damon Runyon Cancer Research Foundation (DRG-2318-18). Peptide synthesis was performed at the Peptide Synthesis Core Facility of the Simpson Querrey Institute for BioNanotechnology at Northwestern University. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

ASJC Scopus subject areas

Analytical Chemistry

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abstract = "G protein-coupled receptors (GPCRs) are the largest class of integral membrane receptors and responsible for transmitting diverse signals in response to extracellular stimuli. Post-translational modifications serve to dictate the subcellular trafficking and function of a GPCR across space and time. Despite significant interest in mapping the diversity of GPCR modification states (proteoforms), technical challenges have hindered this characterization. While advancements in membrane mimetics and mass spectrometry instrumentation have improved analysis, current workflows require large amounts of homogeneous protein, limiting the study of many GPCRs from mammalian sources. Here, we present capillary zone electrophoresis (CZE) as a separation technique for characterizing proteoforms of both intact and partially digested GPCRs. This method allowed for the characterization of multiple proteoforms of both the β2-adrenergic receptor and metabotropic glutamate receptor 2 using low sample volumes and without buffer optimization. Notably, in the case of smaller phosphorylated analytes, CZE can readily separate positional phosphorylation isomers and provide superior fragmentation coverage to conventional reversed phase-liquid chromatography.",

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AU - Janetzko, John

AU - Kelleher, Neil L.

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Capillary Zone Electrophoresis-Mass Spectrometry of Intact G Protein-Coupled Receptors Enables Proteoform Profiling

Abstract

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