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J. Biol. Chem. 280 (7): 5972-5982

© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Proteomic Analysis of in Vivo Phosphorylated Synaptic Proteins*

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Mark O. Collins{ddagger}§, Lu Yu§, Marcelo P. Coba§, Holger Husi{ddagger}, Iain Campuzano¶, Walter P. Blackstock||, Jyoti S. Choudhary§, , and Seth G. N. Grant{ddagger}§**

{ddagger}Division of Neuroscience, University of Edinburgh, Edinburgh EH8 9JZ, United Kingdom, the §Wellcome Trust Sanger Institute, Hinxton CB10 1SA, United Kingdom, Waters Corp., Manchester M22 5PP, United Kingdom, and the ||Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom

Abstract: 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.


Received for publication September 30, 2004. Revision received November 16, 2004.

* This work was spported by the Wellcome Trust (to H. H., J. S. C., L. Y., M. O. C., M. P. C., and S. G. N. G.) and the Biotechnology and Biological Sciences Research Council (to M. O. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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The on-line version of this article (available at http://www.jbc.org) contains an additional figure and four tables.

** To whom correspondence should be addressed: Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire CB10 1SA, United Kingdom. Tel.: 44-1223-494-908; Fax: 44-1233-494-919; E-mail: sg3{at}sanger.ac.uk.


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