Premium
Quantitative phosphoproteomics of depolarization‐dependent protein phosphorylation in nerve terminals
Author(s) -
Larsen Martin R.,
Palmisano Giuseppe,
EngholmKeller Kasper,
Kulej Katarzyna,
Xue Jung,
Graham Mark,
Robinson Phillip J.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.905.2
Subject(s) - phosphoproteomics , exocytosis , phosphorylation , endocytosis , depolarization , chemistry , protein phosphorylation , microbiology and biotechnology , phosphatase , kinase , biochemistry , protein kinase a , biophysics , biology , cell , membrane
Depolarization‐dependent calcium influx activates a number of kinases and phosphatases which result in altered phosphorylation of key proteins in, exocytosis, endocytosis etc. Only a small number of phosphosites in these proteins have been identified to date. Here we have used iTRAQ labeling directly on TiO2 resin combined with the SIMAC strategy (Thingholm TE et al., MCP 2007), Hydrophilic interaction chromatography and high accuracy mass spectrometry to quantitatively assess phosphorylation sites in synaptosomes treated with KCL and low and high levels of 4‐aminopyridine (4AP). Using the new quantitative phosphoproteomics strategy we have successfully identified more than 1500 phosphorylated proteins from highly pure synaptosomes. In these 1500 phosphoproteins we have identified and quantified more than 2500 phosphosites. More than 30 % of all the phosphosites change upon depolarization using either KCL or 4AP. In addition, we have identified 72 kinases and 19 phosphatases in synaptosomes whereof many of those are specifically regulated upon stimulation. Overall we show changes in the phosphorylation status of proteins participating in e.g., transportation of SVs, exocytosis and endocytosis.