Proteomic analysis of in vivo phosphorylated synaptic proteins

被引:261
作者
Collins, MO
Yu, L
Coba, MP
Husi, H
Campuzano, L
Blackstock, WP
Choudhary, JS
Grant, SGN [1 ]
机构
[1] Wellcome Trust Sanger Inst, Hinxton CB10 1SA, Cambs, England
[2] Univ Edinburgh, Div Neurosci, Edinburgh EH8 9JZ, Midlothian, Scotland
[3] Waters Corp, Manchester M22 5PP, Lancs, England
[4] Univ Sheffield, Dept Mol Biol & Biotechnol, Sheffield S10 2TN, S Yorkshire, England
关键词
D O I
10.1074/jbc.M411220200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
In the nervous system, protein phosphorylation is an essential feature of synaptic function. Although protein phosphorylation is known to be important for many synaptic processes and in disease, little is known about global phosphorylation of synaptic proteins. Heterogeneity and low abundance make protein phosphorylation analysis difficult, particularly for mammalian tissue samples. Using a new approach, combining both protein and peptide immobilized metal affinity chromatography and mass spectrometry data acquisition strategies, we have produced the first large scale map of the mouse synapse phosphoproteome. We report over 650 phosphorylation events corresponding to 331 sites (289 have been unambiguously assigned), 92% of which are novel. These represent 79 proteins, half of which are novel phosphoproteins, and include several highly phosphorylated proteins such as MAP1B (33 sites) and Bassoon (30 sites). An additional 149 candidate phosphoproteins were identified by profiling the composition of the protein immobilized metal affinity chromatography enrichment. All major synaptic protein classes were observed, including components of important pre- and postsynaptic complexes as well as low abundance signaling proteins. Bioinformatic and in vitro phosphorylation assays of peptide arrays suggest that a small number of kinases phosphorylate many proteins and that each substrate is phosphorylated by many kinases. These data substantially increase existing knowledge of synapse protein phosphorylation and support a model where the synapse phosphoproteome is functionally organized into a highly interconnected signaling network.
引用
收藏
页码:5972 / 5982
页数:11
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