Journal article
Targeted tandem affinity purification of PSD-95 recovers core postsynaptic complexes and schizophrenia susceptibility proteins
E Fernández, MO Collins, RT Uren, MV Kopanitsa, NH Komiyama, MDR Croning, L Zografos, JD Armstrong, JS Choudhary, SGN Grant
Molecular Systems Biology | WILEY | Published : 2009
DOI: 10.1038/msb.2009.27
Abstract
The molecular complexity of mammalian proteomes demands new methods for mapping the organization of multiprotein complexes. Here, we combine mouse genetics and proteomics to characterize synapse protein complexes and interaction networks. New tandem affinity purification (TAP) tags were fused to the carboxyl terminus of PSD-95 using gene targeting in mice. Homozygous mice showed no detectable abnormalities in PSD-95 expression, subcellular localization or synaptic electrophysiological function. Analysis of multiprotein complexes purified under native conditions by mass spectrometry defined known and new interactors: 118 proteins comprising crucial functional components of synapses, including..
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Funding Acknowledgements
We thank Dr M Pardo for advice on purification conditions and information on cleaved retained proteins, Dr L Yu and S Swamy for support in mass spectrometry analysis, K Porter for tissue perfusion, J Robinson and K Elsegood for mouse colony management, Dr A Enright for protein interaction data, and N Afinowi for the isolation of primary neurons. EF was supported by a Federation of European Biochemistry Societies postdoctoral fellowship; JSC, MOC, MVK, NHK, MDRC and SGNG were supported by the Wellcome Trust; RU was supported by Marie Curie Actions: Research Training Network programs. LZ was supported by the EPSRC/MRC Doctoral Training Centre in Neuroinformatics and Computational Neuroscience.