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Qualitative and quantitative analyses of protein phosphorylation in naive and stimulated mouse synaptosomal preparations


Munton, R P; Tweedie-Cullen, R; Livingstone-Zatchej, M; Weinandy, F; Waidelich, M; Longo, D; Gehrig, P; Potthast, F; Rutishauser, D; Gerrits, B; Panse, C; Schlapbach, R; Mansuy, I M (2007). Qualitative and quantitative analyses of protein phosphorylation in naive and stimulated mouse synaptosomal preparations. Molecular & Cellular Proteomics, 6(2):283-293.

Abstract

Activity-dependent protein phosphorylation is a highly dynamic yet tightly regulated process essential for cellular signaling. Although recognized as critical for neuronal functions, the extent and stoichiometry of phosphorylation in brain cells remain undetermined. In this study, we resolved activity-dependent changes in phosphorylation stoichiometry at specific sites in distinct subcellular compartments of brain cells. Following highly sensitive phosphopeptide enrichment using immobilized metal affinity chromatography and mass spectrometry, we isolated and identified 974 unique phosphorylation sites on 499 proteins, many of which are novel. To further explore the significance of specific phosphorylation sites, we used isobaric peptide labels and determined the absolute quantity of both phosphorylated and non-phosphorylated peptides of candidate phosphoproteins and estimated phosphorylation stoichiometry. The analyses of phosphorylation dynamics using differentially stimulated synaptic terminal preparations revealed activity-dependent changes in phosphorylation stoichiometry of target proteins. Using this method, we were able to differentiate between distinct isoforms of Ca2+/calmodulin-dependent protein kinase (CaMKII) and identify a novel activity-regulated phosphorylation site on the glutamate receptor subunit GluR1. Together these data illustrate that mass spectrometry-based methods can be used to determine activity-dependent changes in phosphorylation stoichiometry on candidate phosphopeptides following large scale phosphoproteome analysis of brain tissue.

Abstract

Activity-dependent protein phosphorylation is a highly dynamic yet tightly regulated process essential for cellular signaling. Although recognized as critical for neuronal functions, the extent and stoichiometry of phosphorylation in brain cells remain undetermined. In this study, we resolved activity-dependent changes in phosphorylation stoichiometry at specific sites in distinct subcellular compartments of brain cells. Following highly sensitive phosphopeptide enrichment using immobilized metal affinity chromatography and mass spectrometry, we isolated and identified 974 unique phosphorylation sites on 499 proteins, many of which are novel. To further explore the significance of specific phosphorylation sites, we used isobaric peptide labels and determined the absolute quantity of both phosphorylated and non-phosphorylated peptides of candidate phosphoproteins and estimated phosphorylation stoichiometry. The analyses of phosphorylation dynamics using differentially stimulated synaptic terminal preparations revealed activity-dependent changes in phosphorylation stoichiometry of target proteins. Using this method, we were able to differentiate between distinct isoforms of Ca2+/calmodulin-dependent protein kinase (CaMKII) and identify a novel activity-regulated phosphorylation site on the glutamate receptor subunit GluR1. Together these data illustrate that mass spectrometry-based methods can be used to determine activity-dependent changes in phosphorylation stoichiometry on candidate phosphopeptides following large scale phosphoproteome analysis of brain tissue.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Functional Genomics Center Zurich
08 University Research Priority Programs > Systems Biology / Functional Genomics
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:February 2007
Deposited On:18 Dec 2009 13:26
Last Modified:06 Dec 2017 22:05
Publisher:American Society for Biochemistry and Molecular Biology
ISSN:1535-9476
Additional Information:This research was originally published in Molecular & Cellular Proteomics. Munton, R P et al. Qualitative and quantitative analyses of protein phosphorylation in naive and stimulated mouse synaptosomal preparations. Molecular & Cellular Proteomics 2007, 6:283-293. © the American Society for Biochemistry and Molecular Biology.
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1074/mcp.M600046-MCP200
PubMed ID:17114649

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