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- Experimental procedures
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Characterization of the composition of the postsynaptic proteome (PSP) provides a framework for understanding the overall organization and function of the synapse in normal and pathological conditions. We have identified 698 proteins from the postsynaptic terminal of mouse CNS synapses using a series of purification strategies and analysis by liquid chromatography tandem mass spectrometry and large-scale immunoblotting. Some 620 proteins were found in purified postsynaptic densities (PSDs), nine in AMPA-receptor immuno-purifications, 100 in isolates using an antibody against the NMDA receptor subunit NR1, and 170 by peptide-affinity purification of complexes with the C-terminus of NR2B. Together, the NR1 and NR2B complexes contain 186 proteins, collectively referred to as membrane-associated guanylate kinase-associated signalling complexes. We extracted data from six other synapse proteome experiments and combined these with our data to provide a consensus on the composition of the PSP. In total, 1124 proteins are present in the PSP, of which 466 were validated by their detection in two or more studies, forming what we have designated the Consensus PSD. These synapse proteome data sets offer a basis for future research in synaptic biology and will provide useful information in brain disease and mental disorder studies.
An important goal of cognitive science is to identify neural mechanisms for processing information. The synapse both transmits information between cells and processes it by detecting specific patterns of neural activity and converting this electrical activity into intracellular biochemical events that change the properties of the neuron (Greengard 2001; Kandel 2001). In recent years it has become clear that synapses, like other cell–cell interactions in metazoans, utilize signal transduction complexes and pathways with a high degree of molecular complexity and cross-talk (Pawson and Nash 2003; Sheng and Kim 2002). A major challenge is to devise strategies that extract emergent physiological properties and simple biological principles from highly complex genomic and proteomic data sets, and shed light on both mechanisms and disease processes. The synapse proteome is a suitable prototype to explore these general issues for several reasons. First, it contains a highly localized set of proteins found in dendritic spines. Second, an important role for signalling complexes and pathways has been established. Third, signalling can be studied using patterns of action potentials and, finally, genetic and pharmacological perturbations result in behavioural changes. However, the molecular complexity and global organization is not well understood.
The neurotransmitter glutamate activates synaptic plasticity primarily via the ionotropic NMDA receptor and metabotropic (mGluR) receptors. This leads to an increased Ca2+ level in the dendritic spine and signal transduction to α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors and other effector mechanisms. The cytoplasmic C-termini of NMDA receptor subunits (NR2A/ɛ1, NR2B/ɛ2) bind PDZ domains of PSD-95, a membrane-associated guanylate kinase (MAGUK) protein. Previous proteomic analysis of NMDA receptor–PSD-95 complexes identified 77 proteins including mGluR receptors, whereas AMPA receptors were found in different complexes (Husi et al. 2000). These complexes are embedded in the postsynaptic terminal of excitatory synapses with other postsynaptic proteins.
Here we present a large-scale proteomic analysis of the postsynaptic proteome (PSP) of mouse brain. We have performed a MS-based analysis of MAGUK-associated signalling complexes (MASCs), AMPA receptor complexes (ARCs) and isolated PSDs, and validated a set of MASC and PSD proteins by large-scale immunoblotting. In addition to generating novel data, we have systematically compiled data from publicly available sources to produce a comprehensive data set of the composition of the PSP. This includes data on the PSD and various subcomponents, namely NMDA receptor complexes (NRCs), ARCs and MASCs.
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- Experimental procedures
- Supporting Information
We have presented a comprehensive analysis of isolated PSDs and embedded multiprotein complexes associated with NMDA and AMPA receptors. We have combined existing synapse proteomic data with that reported here to provide the most complete picture of the PSP to date. To our knowledge, the PSP is one of the most complicated subcellular structures in the eukaryotic cell but, unlike other subcellular structures, the PSP appears to hold a high degree of cellular autonomy. The composition of the PSP in terms of functional protein classes is diverse, with the necessary molecular machinery for not only classical synaptic processes and a myriad of signalling pathways, but for a host of supporting activities such as protein synthesis and degradation, transport and metabolism. Moreover, 10% of PSP proteins are novel and may participate in synaptic processes that are currently unknown.
It is clear that there are a large number of proteins at the postsynaptic membrane that coordinate a wide variety of cellular processes. Is there evidence of functional organization at the postsynaptic membrane? We have observed organization at many levels; from the classes of proteins present at the PSD, the enrichment of scaffolding and signalling domains in PSD proteins, to macromolecular organization in terms of multiprotein complexes. These levels of organization contribute to, or are dependent on, protein–protein interactions and so mapping and understanding protein–protein interactions in the PSP will be very useful. Protein interactions are essential to the functionality of proteomes because proteins rarely act alone, but rather in complexes. Large-scale mapping of protein interactions in the yeast proteome reveals highly complex networks of protein interactions (Fromont-Racine et al. 1997; Gavin et al. 2002). To facilitate analysis of the organization of the PSP at the level of protein interactions we have constructed an in-house database (http//:http://www.PPID.org). This database is a mammalian (mouse, rat, human) Protein–Protein Interaction Database (PPID) describing approximately 8000 biochemically defined protein interactions for more than 2000 proteins. So far we have performed literature mining for PSP protein interactions and have found 650 protein–protein interactions for 281 PSP proteins (of which the majority are MASC proteins). We are using these data to perform network analyses to investigate protein interaction network architecture in the PSP (Grant 2003a). In addition to literature mining, we are performing immuno-affinity purifications on PSP proteins to map additional multiprotein complexes at the synapse.
The large number of proteins found at the PSD, and the presence of many ribosomal, mitochondrial and nuclear proteins, has prompted speculation over the purity of isolated PSD fractions used in proteomic analyses. There are numerous examples of such proteins being validated as true PSD proteins. Some proteins, such as voltage-dependent anion channel 1, which was originally found in the outer mitochondrial membrane, is present in the PSD (Moon et al. 1999). Nuclear proteins such as AIDA-1 A, mKIAA0417 and hnRNP localize to both the nucleus and the PSD (Jordan et al. 2004). It is apparent that many proteins do not exist in discrete locations or indeed possess the same functions in different cellular contexts.
The data presented and compiled in this study represent a draft of an average isolated PSD. The starting material for the analyses incorporated into our draft PSP are from different species (mouse and rat) and from different brain regions (forebrain and whole brain) and encompass hundreds of neuronal cell types. In addition, it is probable that some components of the MASC are interacting with NMDA receptors during their trafficking, localization and internalization. It is now is becoming feasible to compare different types of synapse proteomes. This could be achieved by subtractive proteomics (Andersen et al. 2003), a quantitative approach whereby synaptosomes and isolated PSDs could be isotopically tagged (ICAT or iTRAQ (isotopic tags for relative and obsolute quantification)) and the resulting quantitative MS data would indicate enrichment or depletion of proteins in the PSD compared with synaptosomes. This type of analysis would provide confident assignment of true PSD proteins, highlight contaminating proteins and would validate the PSD localization of proteins currently considered to be restricted to other cellular compartments.
In addition, isolated PSDs or protein complexes from different brain regions or cultured cell types could be compared quantitatively using isotopic labelling. Alternatively, peptides derived from SILAC (stable isotope labelling with amino acids in cell culture)-labelled cells could be used quantitatively to compare the expression of proteins in many tissue samples (Ishihama et al. 2005). Using these approaches, proteins enriched in a particular cell type or brain region would be identified as being potentially more relevant to the physiology of that particular cell or tissue type. It is at this stage that systematic immuno-cytochemistry of these enriched proteins would be feasible and would provide complementary information regarding their specific protein localization at the PSD and throughout the cell type in question. Current projects, such as Gene Expression Nervous System Atlas (GENSAT), in which large-scale in situ hybridization and Bacteriol Artificial Chromosome (BAC) transgenic reporter genes are being used to map gene expression in neuronal cell types, will provide complementary information to the proteomic experiments suggested here.
This draft PSP data set constitutes a candidate gene list for the Genes to Cognition research programme in which the functions of a large number of these genes are being studied by systematic targeted mutations in mice (Grant 2003b). Comprehensive phenotypic analysis of these mutants in terms of biochemistry, electrophysiology, cell biology and behaviour is allowing functional annotation of PSP components. This approach, together with single-nucleotide polymorphism screening in humans for all genes in the PSP, will produce systematic data relevant to synaptic physiology and disease. Establishment of this PSP data set (available at http://www.genes2cognition.org/science/proteomics/synapse.html) is essential for the progression of synapse proteomics and, with new quantitative proteomic strategies that may be applied to activated or diseased states of the synapse, will also provide a benchmark for analysis of dynamic aspects of the synapse proteome.