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.
Highlights
Activity-dependent protein phosphorylation is a highly dynamic yet tightly regulated process essential for cellular signaling
Selective, and reproducible phosphopeptide enrichment method based on strong cation exchange chromatography (SCX) and IMAC, we identified multiple candidate phosphorylation sites in proteolytic digests of synaptic membranes, postsynaptic densities (PSDs), and synaptic vesicles
Streamlined High Throughput Identification of Phosphorylation Sites—Peptides from proteolytic digests of synaptic membranes, PSD, or synaptic vesicles were first fractionated by charge using SCX and subjected to a second phosphopeptide-specific enrichment using IMAC
Summary
Activity-dependent protein phosphorylation is a highly dynamic yet tightly regulated process essential for cellular signaling. 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. 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. Several studies have examined subcellular neural phosphoproteomes [11,12,13,14,15,16] the dynamics and stoichiometry of protein phosphorylation in the brain remain unstudied
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