Qualitative and quantitative analyses of protein phosphorylation in naive and stimulated mouse synaptosomal preparations.
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.
Munton
Richard P
RP
Brain Research Institute, Medical Faculty of the University of Zürich, Switzerland.
Tweedie-Cullen
Ry
R
Livingstone-Zatchej
Magdalena
M
Weinandy
Franziska
F
Waidelich
Marc
M
Longo
Davide
D
Gehrig
Peter
P
Potthast
Frank
F
Rutishauser
Dorothea
D
Gerrits
Bertran
B
Panse
Christian
C
Schlapbach
Ralph
R
Mansuy
Isabelle M
IM
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D016000:Cluster Analysis; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
D010455:Peptides