Composition of the Synaptic PSD-95 Complex
2007; Elsevier BV; Volume: 6; Issue: 10 Linguagem: Inglês
10.1074/mcp.m700040-mcp200
ISSN1535-9484
AutoresAyṣe Döṣemeci, Anthony J. Makusky, Ewa Jankowska‐Stephens, Xiaoyu Yang, Douglas J. Slotta, Sanford P. Markey,
Tópico(s)Photoreceptor and optogenetics research
ResumoPostsynaptic density protein 95 (PSD-95), a specialized scaffold protein with multiple protein interaction domains, forms the backbone of an extensive postsynaptic protein complex that organizes receptors and signal transduction molecules at the synaptic contact zone. Large, detergent-insoluble PSD-95-based postsynaptic complexes can be affinity-purified from conventional PSD fractions using magnetic beads coated with a PSD-95 antibody. In the present study purified PSD-95 complexes were analyzed by LC/MS/MS. A semiquantitative measure of the relative abundances of proteins in the purified PSD-95 complexes and the parent PSD fraction was estimated based on the cumulative ion current intensities of corresponding peptides. The affinity-purified preparation was largely depleted of presynaptic proteins, spectrin, intermediate filaments, and other contaminants prominent in the parent PSD fraction. We identified 525 of the proteins previously reported in parent PSD fractions, but only 288 of these were detected after affinity purification. We discuss 26 proteins that are major components in the PSD-95 complex based upon abundance ranking and affinity co-purification with PSD-95. This subset represents a minimal list of constituent proteins of the PSD-95 complex and includes, in addition to the specialized scaffolds and N-methyl-d-aspartate (NMDA) receptors, an abundance of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, small G-protein regulators, cell adhesion molecules, and hypothetical proteins. The identification of two Arf regulators, BRAG1 and BRAG2b, as co-purifying components of the complex implies pivotal functions in spine plasticity such as the reorganization of the actin cytoskeleton and insertion and retrieval of proteins to and from the plasma membrane. Another co-purifying protein (Q8BZM2) with two sterile α motif domains may represent a novel structural core element of the PSD. Postsynaptic density protein 95 (PSD-95), a specialized scaffold protein with multiple protein interaction domains, forms the backbone of an extensive postsynaptic protein complex that organizes receptors and signal transduction molecules at the synaptic contact zone. Large, detergent-insoluble PSD-95-based postsynaptic complexes can be affinity-purified from conventional PSD fractions using magnetic beads coated with a PSD-95 antibody. In the present study purified PSD-95 complexes were analyzed by LC/MS/MS. A semiquantitative measure of the relative abundances of proteins in the purified PSD-95 complexes and the parent PSD fraction was estimated based on the cumulative ion current intensities of corresponding peptides. The affinity-purified preparation was largely depleted of presynaptic proteins, spectrin, intermediate filaments, and other contaminants prominent in the parent PSD fraction. We identified 525 of the proteins previously reported in parent PSD fractions, but only 288 of these were detected after affinity purification. We discuss 26 proteins that are major components in the PSD-95 complex based upon abundance ranking and affinity co-purification with PSD-95. This subset represents a minimal list of constituent proteins of the PSD-95 complex and includes, in addition to the specialized scaffolds and N-methyl-d-aspartate (NMDA) receptors, an abundance of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, small G-protein regulators, cell adhesion molecules, and hypothetical proteins. The identification of two Arf regulators, BRAG1 and BRAG2b, as co-purifying components of the complex implies pivotal functions in spine plasticity such as the reorganization of the actin cytoskeleton and insertion and retrieval of proteins to and from the plasma membrane. Another co-purifying protein (Q8BZM2) with two sterile α motif domains may represent a novel structural core element of the PSD. The postsynaptic density (PSD) 1The abbreviations used are: PSD, postsynaptic density; NMDA, N-methyl-d-aspartate; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; Arf, ADP-ribosylation factor; SAM, sterile α motif; CaMKII, calcium/calmodulin-dependent protein kinase II; EM, electron microscopy; SAPAP, SAP90/PSD-95-associated protein; TARP, transmembrane AMPA receptor regulatory protein; BRAG, brefeldin A-resistant ArfGEF; GEF, guanine nucleotide exchange factor; GAP, GTPase-activating protein. 1The abbreviations used are: PSD, postsynaptic density; NMDA, N-methyl-d-aspartate; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; Arf, ADP-ribosylation factor; SAM, sterile α motif; CaMKII, calcium/calmodulin-dependent protein kinase II; EM, electron microscopy; SAPAP, SAP90/PSD-95-associated protein; TARP, transmembrane AMPA receptor regulatory protein; BRAG, brefeldin A-resistant ArfGEF; GEF, guanine nucleotide exchange factor; GAP, GTPase-activating protein. is a disk-shaped protein complex lining the postsynaptic membrane. In a recent study its total mass was estimated to be around 1 million kDa (1Chen X. Vinade L. Leapman R.D. Petersen J.D. Nakagawa T. Phillips T.M. Sheng M. Reese T.S. Mass of the postsynaptic density and enumeration of three key molecules.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 11551-11556Crossref PubMed Scopus (174) Google Scholar). The function of this massive protein complex appears to be anchoring and organizing postsynaptic neurotransmitter receptors and corresponding signaling molecules at the active zone. Thus, it is expected that the extent and type of the postsynaptic response to neurotransmitter release will largely depend on the molecular composition and organization of the PSD.The first tentative identification of PSD components was done in the late 1970s when several laboratories developed the methodology to isolate PSD fractions and started analyzing them by biochemical methods (2Cohen R.S. Blomberg F. Berzins K. Siekevitz P. The structure of postsynaptic densities isolated from dog cerebral cortex. I. Overall morphology and protein composition.J. Cell Biol. 1977; 74: 181-203Crossref PubMed Scopus (383) Google Scholar, 3Matus A.I. Taff-Jones D.H. Morphology and molecular composition of isolated postsynaptic junctional structures.Proc. R. Soc. Lond. B Biol. Sci. 1978; 203: 135-151Crossref PubMed Scopus (90) Google Scholar). The general strategy, still applied today, was treatment of synaptosomal fractions with detergents that solubilize membranes but leave the PSD relatively intact and subsequent separation of membrane-free PSDs by further centrifugation. Analysis of PSD fractions continued to reveal additional putative PSD components in later years (4Walsh M.J. Kuruc N. The postsynaptic density: constituent and associated proteins characterized by electrophoresis, immunoblotting, and peptide sequencing.J. 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Proteome Res. 2005; 4: 725-733Crossref PubMed Scopus (69) Google Scholar, 11Collins M.O. Yu L. Coba M.P. Husi H. Campuzano I. Blackstock W.P. Choudhary J.S. Grant S.G. Proteomic analysis of in vivo phosphorylated synaptic proteins.J. Biol. Chem. 2005; 280: 5972-5982Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 12Trinidad J.C. Thalhammer A. Specht C.G. Schoepfer R. Burlingame A.L. Phosphorylation state of postsynaptic density proteins.J. Neurochem. 2005; 92: 1306-1316Crossref PubMed Scopus (67) Google Scholar, 13Trinidad J.C. Specht C.G. Thalhammer A. Schoepfer R. Burlingame A.L. Comprehensive identification of phosphorylation sites in postsynaptic density preparations.Mol. Cell. Proteomics. 2006; 5: 914-922Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 14Cheng D. Hoogenraad C.C. Rush J. Ramm E. Schlager M.A. Duong D.M. Xu P. Wijayawardana S.R. Hanfelt J. Nakagawa T. Sheng M. Peng J. Relative and absolute quantification of postsynaptic density proteome isolated from rat forebrain and cerebellum.Mol. Cell. Proteomics. 2006; 5: 1158-1170Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). The development of two-hybrid screens and other methods to determine binding partners of already identified components added important complementary approaches (for a review, see Ref. 15Kim E. Sheng M. PDZ domain proteins of synapses.Nat. Rev. Neurosci. 2004; 5: 771-781Crossref PubMed Scopus (1210) Google Scholar).The global proteomics analysis of isolated PSDs remains a crucial first step in the elucidation of the molecular structure of the PSD. Indeed the strategy constitutes a relatively simple and convenient way for the identification of hundreds of proteins in a single run: one of the most recent studies identified a total of 1264 proteins (13Trinidad J.C. Specht C.G. Thalhammer A. Schoepfer R. Burlingame A.L. Comprehensive identification of phosphorylation sites in postsynaptic density preparations.Mol. Cell. Proteomics. 2006; 5: 914-922Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Also unlike immunological approaches, the strategy is not based on any a priori notion of PSD constituents and therefore can reveal hitherto unsuspected elements. A recent study (16Collins M.O. Husi H. Yu L. Brandon J.M. Anderson C.N. Blackstock W.P. Choudhary J.S. Grant S.G. Molecular characterization and comparison of the components and multiprotein complexes in the postsynaptic proteome.J. Neurochem. 2006; 97: 16-23Crossref PubMed Scopus (341) Google Scholar) integrated data from seven proteomics studies (5Walikonis R.S. Jensen O.N. Mann M. Provance D.W.J. Mercer J.A. Kennedy M.B. Identification of proteins in the postsynaptic density fraction by mass spectrometry.J. Neurosci. 2000; 20: 4069-4080Crossref PubMed Google Scholar, 6Jordan B.A. Fernholz B.D. Boussac M. Xu C. Grigorean G. Ziff E.B. Neubert T.A. Identification and verification of novel rodent postsynaptic density proteins.Mol. Cell. Proteomics. 2004; 3: 857-871Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 7Li K.W. Hornshaw M.P. Van Der Schors R.C. Watson R. Tate S. Casetta B. Jimenez C.R. Gouwenberg Y. Gundelfinger E.D. Smalla K.H. Smit A.B. Proteomics analysis of rat brain postsynaptic density. Implications of the diverse protein functional groups for the integration of synaptic physiology.J. Biol. Chem. 2004; 279: 987-1002Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 8Yoshimura Y. Yamauchi Y. Shinkawa T. Taoka M. Donai H. Takahashi N. Isobe T. Yamauchi T. Molecular constituents of the postsynaptic density fraction revealed by proteomic analysis using multidimensional liquid chromatography-tandem mass spectrometry.J. Neurochem. 2004; 88: 759-768Crossref PubMed Scopus (178) Google Scholar, 9Peng J. Kim M.J. Cheng D. Duong D.M. Gygi S.P. Sheng M. Semiquantitative proteomic analysis of rat forebrain postsynaptic density fractions by mass spectrometry.J. Biol. Chem. 2004; 279: 21003-21011Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 17Satoh K. Takeuchi M. Oda Y. Deguchi-Tawarada M. Sakamoto Y. Matsubara K. Nagasu T. Takai Y. Identification of activity-regulated proteins in the postsynaptic density fraction.Genes Cells. 2002; 7: 187-197Crossref PubMed Scopus (75) Google Scholar) and other literature on the analysis of PSD fractions. Collins et al. (16Collins M.O. Husi H. Yu L. Brandon J.M. Anderson C.N. Blackstock W.P. Choudhary J.S. Grant S.G. Molecular characterization and comparison of the components and multiprotein complexes in the postsynaptic proteome.J. Neurochem. 2006; 97: 16-23Crossref PubMed Scopus (341) Google Scholar) report that altogether 1124 proteins were identified in these seven studies. However, 58% of the proteins were detected in only one study, raising the possibility of a high rate of false positives. The authors also compiled a "consensus PSD" list of 467 proteins that were identified in at least two of the studies, thus reducing the probability of false positives linked to individual protocols.Although the 467 proteins in the consensus PSD list have a better probability of being genuine PSD components, detection in multiple studies does not necessarily prove that they are. In fact, there seem to be only a handful of proteins that were identified in all seven of the studies (Ref. 16Collins M.O. Husi H. Yu L. Brandon J.M. Anderson C.N. Blackstock W.P. Choudhary J.S. Grant S.G. Molecular characterization and comparison of the components and multiprotein complexes in the postsynaptic proteome.J. Neurochem. 2006; 97: 16-23Crossref PubMed Scopus (341) Google Scholar and Supplemental Tables S2 and S3 in Ref. 16Collins M.O. Husi H. Yu L. Brandon J.M. Anderson C.N. Blackstock W.P. Choudhary J.S. Grant S.G. Molecular characterization and comparison of the components and multiprotein complexes in the postsynaptic proteome.J. Neurochem. 2006; 97: 16-23Crossref PubMed Scopus (341) Google Scholar), and this high consensus group includes, in addition to the expected PSD components such as PSD-95, homer, and CaMKII, likely contaminants such as synapsins and intermediate filaments. Moreover because the probability of detection of a protein in a mixture increases with its relative abundance, it is likely that the contaminants in this group are among the most abundant in PSD fractions.Detection of the same contaminants in all proteomics studies suggests a systemic contamination problem in PSD fractions in general. The most widely applied protocol for the preparation of PSD fractions is the one originally developed by Carlin et al. (18Carlin R.K. Grab D.J. Cohen R.S. Siekevitz P. Isolation and characterization of postsynaptic densities from various brain regions: enrichment of different types of postsynaptic densities.J. Cell Biol. 1980; 86: 831-845Crossref PubMed Scopus (599) Google Scholar) and uses the relatively mild detergent Triton X-100. Because Triton X-100 is mild, it appears to cause minimal loss of protein from the PSD as judged from morphological criteria. On the other hand, many proteins that are present in the synaptosomal fraction but are not part of the PSD also appear to be resistant to Triton X-100. Indeed electron microscopy (EM) analyses of PSD fractions prepared by this method reveal various particulate contaminants including filamentous material and other protein complexes (19Dosemeci A. Reese T.S. Petersen J. Tao-Cheng J.H. A novel particulate form of Ca2+/calmodulin-dependent [correction of Ca2+/CaMKII-dependent] protein kinase II in neurons.J. Neurosci. 2000; 20: 3076-3084Crossref PubMed Google Scholar, 20Vinade L. Chang M. Schlief M.L. Petersen J.D. Reese T.S. Tao-Cheng J.H. Dosemeci A. Affinity purification of PSD-95-containing postsynaptic complexes.J. Neurochem. 2003; 87: 1255-1261Crossref PubMed Scopus (26) Google Scholar). Some of the filamentous materials that are not associated with the PSD have been identified as neurofilaments and spectrin (20Vinade L. Chang M. Schlief M.L. Petersen J.D. Reese T.S. Tao-Cheng J.H. Dosemeci A. Affinity purification of PSD-95-containing postsynaptic complexes.J. Neurochem. 2003; 87: 1255-1261Crossref PubMed Scopus (26) Google Scholar).Altogether these considerations indicate the need for a purer preparation as a basis for the identification of PSD components. The use of a stronger detergent such as N-laurylsarcosinate (21Cho K.O. Hunt C.A. Kennedy M.B. The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein.Neuron. 1992; 9: 929-942Abstract Full Text PDF PubMed Scopus (1001) Google Scholar) eliminates certain contaminants but also appears to dissociate some genuine PSD elements as well. In addition, certain contaminants such as the so-called "CaMKII clusters" that are resistant to the detergent become enriched in the N-laurylsarcosinate-derived PSD fraction (19Dosemeci A. Reese T.S. Petersen J. Tao-Cheng J.H. A novel particulate form of Ca2+/calmodulin-dependent [correction of Ca2+/CaMKII-dependent] protein kinase II in neurons.J. Neurosci. 2000; 20: 3076-3084Crossref PubMed Google Scholar). On the other hand, because most particulate contaminants in the PSD fraction are membrane-free protein complexes like the PSDs themselves, they are expected to be of the same density as PSDs, excluding the possibility of further fractionation by conventional centrifugation-based techniques.Affinity-based separation techniques targeting ubiquitous PSD components constitute an orthogonal approach for the isolation of postsynaptic protein complexes, a strategy expected to avoid the problem of detergent-insoluble particulate material contamination prevalent in conventional density-based PSD preparations. Using this approach the isolation of NMDA receptor and PSD-95 complexes from whole cell deoxycholate extracts has been reported (22Husi H. Ward M.A. Choudhary J.S. Blackstock W.P. Grant S.G. Proteomic analysis of NMDA receptor-adhesion protein signaling complexes.Nat. Neurosci. 2000; 3: 661-669Crossref PubMed Scopus (1017) Google Scholar, 23Husi H. Grant S.G. Isolation of 2000-kDa complexes of N-methyl-d-aspartate receptor and postsynaptic density 95 from mouse brain.J. Neurochem. 2001; 77: 281-291Crossref PubMed Scopus (100) Google Scholar). However, because these preparations are from detergent-solubilized starting material, isolated complexes tend to be small. Indeed the molecular mass of NMDA receptor complexes was estimated to be around 2000 kDa, which is about 3 orders of magnitude smaller than the whole PSD complex. Thus, the preparation presumably contains receptor complexes that become dissociated from the bigger PSD complex upon treatment with the relatively strong detergent deoxycholate. In addition, because whole cell extracts are used as starting material, complexes that originate from extrasynaptic/intracellular pools are expected to co-purify with complexes from the PSD. These may include extrasynaptic receptors and transport packages.We evaluated an affinity-based strategy for the isolation of large PSD-95-containing complexes from the Triton X-100-derived PSD fraction (20Vinade L. Chang M. Schlief M.L. Petersen J.D. Reese T.S. Tao-Cheng J.H. Dosemeci A. Affinity purification of PSD-95-containing postsynaptic complexes.J. Neurochem. 2003; 87: 1255-1261Crossref PubMed Scopus (26) Google Scholar). The strategy aims to minimize the contribution from extrasynaptic/intracellular PSD-95 pools by starting with a PSD-enriched fraction from which such non-synaptic complexes have been largely eliminated. Importantly by using magnetic beads coated with a PSD-95 antibody, large, insoluble complexes are separated without the use of additional detergents to solubilize particulates.Postsynaptic PSD-95 complexes are likely to represent the bulk of the PSD observed by EM in intact cells. Indeed PSD-95 is able to interact with a large number of proteins through its three PDZ domains, an SH3 domain, and a guanylate kinase domain and also can multimerize to form an extended scaffold (for a review, see Ref. 15Kim E. Sheng M. PDZ domain proteins of synapses.Nat. Rev. Neurosci. 2004; 5: 771-781Crossref PubMed Scopus (1210) Google Scholar). Moreover proteins that bind PSD-95 in turn interact with yet other postsynaptic components, thus extending the network. The complexes isolated using PSD-95 antibody-coated magnetic beads resemble in situ PSDs (20Vinade L. Chang M. Schlief M.L. Petersen J.D. Reese T.S. Tao-Cheng J.H. Dosemeci A. Affinity purification of PSD-95-containing postsynaptic complexes.J. Neurochem. 2003; 87: 1255-1261Crossref PubMed Scopus (26) Google Scholar), suggesting that most if not all of the original components have been retained. These considerations suggested that analysis of the isolated PSD-95 complexes would identify most if not all of the components of the in situ PSD.In the present study, isolated PSD-95 complexes and the parent PSD fraction were analyzed in parallel by liquid chromatography coupled to tandem mass spectrometry. For each fraction, identified proteins were ranked according to cumulative ion current intensities of corresponding peptides as a semiquantitative measure of relative abundance. Comparison of the ranks of proteins in the parent and affinity-purified preparations allowed identification of those that co-purify with PSD-95 and, importantly, those that are likely contaminants.RESULTSPSD-95 complexes were affinity-purified from the conventional PSD fraction using magnetic beads coated with an antibody against the core PSD protein PSD-95. Parent and affinity-purified samples were separated by one-dimensional SDS gel electrophoresis and analyzed in parallel. The control sample (Fig. 1, Lane 3) shows no visible band except the secondary antibody used to coat the magnetic beads, indicating the absence of nonspecific binding. The protein profiles of the parent PSD fraction (Fig. 1, Lane 1) and of the affinity-purified PSD-95 complex (Fig. 1, Lane 2) are different, indicating removal of several proteins during purification.Gel lanes in their entirety were cut into 40 fractions, digested with trypsin, and analyzed by LC/MS/MS. The resulting mass spectra were assigned probable peptide sequences using the Mascot search engine (Matrix Sciences). For these studies, the Swiss-Prot mammalian protein reference library was selected, and peptides with Mascot Ion Scores exceeding their Identity Scores were analyzed and grouped using DBParser 2.0. This software applies parsimony analysis to reduce the thousands of identified peptides to a minimal protein list (25). Only proteins from the minimal protein list were considered for further evaluation (Table I, Table II, Table III, Table IV). The results from the two preparations include protein assignments from homologous mammalian proteins other than rat as indicated in the tables of results.Table I50 top ranking of proteins in the affinity-purified PSD-95 complexAffinity purification rankParent rankProtein (family) common nameUniProt KB/Swiss-Prot entryUnique peptides11CaMKIIKCC2A_RAT924PSD-95DLG4_RAT2533β-TubulinTBB5_RAT1647SynGAPSYGP1_RAT2452α-TubulinQ5XIF6_RAT10610PSD-93DLG2_RAT16727GluR2GRIA2_RAT2486ActinACTB_RAT6923Shank3SHAN3_RAT251022BRAG1 (KIAA0522)O60275_HUMAN201117HypotheticalQ8BZM2_MOUSE91248GluR3GRIA3_RAT131335HomerHOME1_RAT101419Shank1SHAN1_RAT241526Shank2SHAN2_RAT181614IRSp53BAIP2_RAT121749BRAG2bQ6DN90_HUMAN41861GluR1GRIA1_RAT71924Densin 180LRRC7_RAT132013α-CateninQ5R416_PONPY132120Glutamine synthetaseGLNA_RAT72265SAPAP4DLGP4_RAT42344SAPAP1DLGP1_RAT82429ADP/ATP translocaseADT1_RAT62547SAPAP3DLGP3_MOUSE82645NMDAR2BNMDE2_RAT132716Myosin 10MYH10_RAT132866SAPAP2DLGP2_RAT62932PP1PP1A_RAT73076NeuroliginNLGN3_RAT73187Hypothetical (FAM81A)Q8TBF8_HUMAN532>93GLUR4GRIA4_MOUSE63384CylindromatosisQ66H62_RAT83430PKC-γKPCG_RAT53512SNIPSNIP_RAT6369PlectinPLEC1_RAT233725Heat shock cognate 71HSP7C_RAT83862NMDAR2AO08948_RAT83937SAP97DLG1_HUMAN44036SAP102DLG3_RAT54192Leu-rich repeat …LRTM1_HUMAN24259Hypothetical (FLJ35778)Q8NA73_HUMAN34315VDAC1VDAC1_RAT74438CitronQ9QX19_RAT64560NMDAR1Q62648_RAT54658TARPCCG8_RAT24718α-ActininQ6GMN8_RAT74828G3PG3P_RAT64941KalirinHAPIP_RAT125069CPG2 proteinQ63128_RAT5 Open table in a new tab Table II50 top ranking of proteins in the conventional (parent) PSD fractionParent rankAffinity purification rankProtein (family) common nameUniPost KB/Swiss-Prot entryUnique peptides11CaMKIIKCC2A_RAT1125α-TubulinQ5XIF6_RAT1333β-TubulinTBB5_RAT1542PSD-95DLG4_RAT24559α-SpectrinSPTA2_RAT5268ActinACTB_RAT774SynGAPSYGP1_RAT27869BassoonBSN_RAT38936PlectinPLEC1_RAT60106PSD-93DLG2_RAT151167α-InternexinAINX_RAT111235SNIPSNIP_RAT181320α-CateninQ5R416_PONPY191416IRSp53BAIP2_RAT141543VDAC1VDAC1_RAT131627Myosin 10MYH10_RAT221711Hypothetical (SAM/PTB)Q8BZM2_MOUSE111847α-ActininQ6GMN8_RAT281914Shank1SHAN1_RAT172021Glutamine synthetaseGLNA_RAT72174Synapsin 2SYN2_RAT162210BRAG1 (KIAA0522)O60275_HUMAN14239Shank3SHAN3_RAT142419Densin 180LRRC7_RAT152537Heat shock cognate 71HSP7C_RAT122615Shank2SHAN2_RAT11277GluR2GRIA2_RAT92848G3PG3P_RAT72924ADP/ATP translocaseADT1_RAT83034PKC-γKPCG_RAT83171ERC protein 2ERC2_RAT123229PP1PP1A_RAT103352β-CateninCTNB1_HUMAN113473VDAC2VDAC2_RAT83513HomerHOME1_RAT93640SAP102DLG3_RAT113739SAP97DLG1_HUMAN33844CitronQ9QX19_RAT73960GAPDHQ8K417_SIGHI44056ArgBP2O35413_RAT84149KalirinHAPIP_RAT114263N-cadherinCADH2_RAT64362VDAC3Q6GSZ1_RAT64423SAPAP1DLGP1_RAT84526NMDAR2BNMDE2_RAT74661Fructose bisphosphate aldolaseALDOA_RABIT24725SAPAP3DLGP3_MOUSE104812GluR3GRIA3_RAT64917BRAG2bQ6DN90_HUMAN65066Mitochondrial glutamate carrierGHC1_MOUSE6 Open table in a new tab Table IIIProteins co-purifying with PSD-95Protein (family) nameUniProt KB/Swiss-Prot entryChange in rank (parent/affinity-purified)Normalized ion current intensity ratio (purified/parent)Glutamate receptors GluR2 (AMPAR)GRIA2_RAT27/7>1 GluR3 (AMPAR)GRIA3_RAT48/12>1 GluR1 (AMPAR)GRIA1_RAT61/18>1 GluR4 (AMPAR)GRIA4_RAT>93/32>1 NMDAR2BNMDE2_RAT45/260.55 NMDAR2AO08948_RAT62/38>1 NMDAR1Q62648_RAT60/450.74Scaffolds PSD-95DLG4_RAT4/21.00 PSD-93DLG2_RAT10/60.67 Shank3SHAN3_RAT23/9>1 Hypothetical (SAM/PTB)Q8BZM2_MOUSE17/110.62 HomerHOME1_RAT35/13>1 Shank2SHAN2_RAT26/150.75 SAPAP4DLGP4_RAT65/22>1 SAPAP1DLGP1_RAT44/230.59 SAPAP2DLGP2_RAT66/28>1G-protein regulators SynGAPSYGP1_RAT7/40.64 BRAG1 (KIAA0522)O60275_HUMAN22/10>1 BRAG2bQ6DN90_HUMAN49/17>1Other NeuroliginNLGN3_RAT76/30>1 Hypothetical (FAM81A)Q8TBF8_HUMAN87/31>1 CylindromatosisQ66H62_RAT84/33>1 Leu-rich repeat …LRTM1_HUMAN92/41>1 Hypothetical (FLJ35778)Q8NA73_HUMAN59/420.73 TARP (γ8)CCG8_RAT58/460.62 CPG2 proteinQ63128_RAT69/500.95 Open table in a new tab Table IVProteins greatly reduced/depleted in the affinity-purified PSD-95 complexProtein (family) nameUniProt KB/Swiss-Prot entryChange in rank (parent/purified)Normalized ion current intensity ratio (purified/parent)Presynaptic proteins BassoonBSN_RAT8/690.01 SNIPSNIP_RAT12/350.06 Synapsin 2SYN2_RAT21/740.02 ERC protein 2ERC2_RAT31/710.04 Synapsin 1SYN1_RATNA/aProteins identified with two or more peptides in parent fraction only.0.00 PiccoloPCLO_RATNA/aProteins identified with two or more peptides in parent fraction only.0.00 RIMRIMS_RATNA/aProteins identified with two or more peptides in parent fraction only.0.00Cytoskeletal elements α-SpectrinSPTA2_RAT5/590.01 PlectinPLEC1_RAT9/360.04 α-InternexinAINX_RAT11/670.01 β-SpectrinSPTN2_RATNA/aProteins identified with two or more peptides in parent fraction only.0.00 Neurofilament-LNFL_RATNA/aProteins identified with two or more peptides in parent fraction only.0.00 Neurofilament-MNFM_RATNA/aProteins identified with two or more peptides in parent fraction only.0.00Different organelle/cell type VDAC1 (mitochondrial)VDAC1_RAT15/430.06 VDAC2 (mitochondrial)VDAC2_RAT34/730.04 Myelin basic proteinMBP_RATNA/aProteins identified with two or more peptides in parent fraction only.0.00 Ribosomal proteinsRLA0_RATNA/aProteins identified with two or more peptides in parent fraction only.0.00Other α-ActininQ6GMN8_RAT18/470.06 SynaptopodinSYNPO_RATNA/aProteins identified with two or more peptides in parent fraction only.0.02 β-CateninCTNB1_HUMAN33/520.09 N-cadherinCADH2_RAT42/630.09a Proteins identified with two or more peptides in parent fraction only. Open table in a new tab Altogether three sets of samples were analyzed, corresponding to two independent parent and affinity-purified preparations. The concatenated parent (from 133 LC/MS/MS runs) and affinity-purified (125 LC/MS/MS runs) files correspond to summed and integrated analyses. Identified proteins were ranked according to summed ion current intensities of their constituent peptides as a relative abundance index. DBParser 3.0 extracts retention time and peak intensity from raw data based on the precursor mass identified by the search engine as described previously for ion trap LC/MS/MS data (26Chelius D. Bondarenko P.V. Quantitative profiling of proteins in complex mixtures using liquid chromatography and mass spectrometry.J. Proteome Res. 2
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