Proteomics Analysis of Rat Brain Postsynaptic Density
2004; Elsevier BV; Volume: 279; Issue: 2 Linguagem: Inglês
10.1074/jbc.m303116200
ISSN1083-351X
AutoresKa Wan Li, Martin Hornshaw, Roel C. van der Schors, Rod B. Watson, Stephen Tate, Bruno Casetta, Connie R. Jiménez, Yvonne Gouwenberg, Eckart D. Gundelfinger, Karl‐Heinz Smalla, August B. Smit,
Tópico(s)Cellular transport and secretion
ResumoThe postsynaptic density contains multiple protein complexes that together relay the presynaptic neurotransmitter input to the activation of the postsynaptic neuron. In the present study we took two independent proteome approaches for the characterization of the protein complement of the postsynaptic density, namely 1) two-dimensional gel electrophoresis separation of proteins in conjunction with mass spectrometry to identify the tryptic peptides of the protein spots and 2) isolation of the trypsin-digested sample that was labeled with isotope-coded affinity tag, followed by liquid chromatography-tandem mass spectrometry for the partial separation and identification of the peptides, respectively. Functional grouping of the identified proteins indicates that the postsynaptic density is a structurally and functionally complex organelle that may be involved in a broad range of synaptic activities. These proteins include the receptors and ion channels for glutamate neurotransmission, proteins for maintenance and modulation of synaptic architecture, sorting and trafficking of membrane proteins, generation of anaerobic energy, scaffolding and signaling, local protein synthesis, and correct protein folding and breakdown of synaptic proteins. Together, these results imply that the postsynaptic density may have the ability to function (semi-) autonomously and may direct various cellular functions in order to integrate synaptic physiology. The postsynaptic density contains multiple protein complexes that together relay the presynaptic neurotransmitter input to the activation of the postsynaptic neuron. In the present study we took two independent proteome approaches for the characterization of the protein complement of the postsynaptic density, namely 1) two-dimensional gel electrophoresis separation of proteins in conjunction with mass spectrometry to identify the tryptic peptides of the protein spots and 2) isolation of the trypsin-digested sample that was labeled with isotope-coded affinity tag, followed by liquid chromatography-tandem mass spectrometry for the partial separation and identification of the peptides, respectively. Functional grouping of the identified proteins indicates that the postsynaptic density is a structurally and functionally complex organelle that may be involved in a broad range of synaptic activities. These proteins include the receptors and ion channels for glutamate neurotransmission, proteins for maintenance and modulation of synaptic architecture, sorting and trafficking of membrane proteins, generation of anaerobic energy, scaffolding and signaling, local protein synthesis, and correct protein folding and breakdown of synaptic proteins. Together, these results imply that the postsynaptic density may have the ability to function (semi-) autonomously and may direct various cellular functions in order to integrate synaptic physiology. The majority of excitatory neurotransmission in the brain occurs via glutamatergic synapses. In the presynaptic element of the synapse, specialized secretion machinery determines the activity-dependent membrane fusion of glutamate-containing vesicles and the release of transmitter into the synaptic cleft. In the postsynaptic element, glutamate receptors and downstream signal transduction are organized by the protein assembly of the postsynaptic density (PSD). 1The abbreviations used are: PSD, postsynaptic density; CaMKII, calcium/calmodulin-dependent protein kinase II; CHIP, C terminus of HSP70 interaction protein; EF-1α, eukaryotic elongation factor-1 α; Hsp, heat shock protein; ICAT, isotope-coded affinity tag; IF-4, eukaryotic initiation factor-4; LC, liquid chromatography; MALDI-TOF/TOF® MS, matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometer; NMDA receptor, N-methyl-d-aspartic acid receptor; PMF, peptide mass fingerprint; SAP, synapse-associated protein; Ub-Pr, ubiquititation/proteasome; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid. Both the presynaptic release machinery and the PSD are electron-dense assemblies (1.Ziff E.B. Neuron. 1997; 19: 1163-1174Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 2.Dresbach T. Qualmann B. Kessels M.M. Garner C.C. Gundelfinger E.D. Cell. Mol. Life Sci. 2001; 58: 94-116Crossref PubMed Scopus (163) Google Scholar), in which proteins are thought to be organized into distinct functional complexes (3.Kennedy M.B. Science. 2000; 290: 750-754Crossref PubMed Scopus (663) Google Scholar, 4.Husi H. Ward M.A. Choudhary J.S. Blackstock W.P. Grant S.G. Nat. Neurosci. 2000; 3: 661-669Crossref PubMed Scopus (1038) Google Scholar, 5.Husi, H., Choudhary, J., Blackstock, W., and Grant, S. G. (2002) FENS Forum 2002 in Paris, Abstr. 112.11Google Scholar) that may be dynamically regulated by neuronal activity (6.Malinow R. Malenka R.C. Annu. Rev. Neurosci. 2002; 25: 103-126Crossref PubMed Scopus (2093) Google Scholar, 7.Yuste R. Bonhoeffer T. Annu. Rev. Neurosci. 2001; 24: 1071-1089Crossref PubMed Scopus (1002) Google Scholar, 8.Huntley G.W. Benson D.L. Colman D.R. Cell. 2002; 108: 1-4Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The modulation of this molecular architecture of the synapse is at the basis of synaptic plasticity (6.Malinow R. Malenka R.C. Annu. Rev. Neurosci. 2002; 25: 103-126Crossref PubMed Scopus (2093) Google Scholar, 7.Yuste R. Bonhoeffer T. Annu. Rev. Neurosci. 2001; 24: 1071-1089Crossref PubMed Scopus (1002) Google Scholar, 8.Huntley G.W. Benson D.L. Colman D.R. Cell. 2002; 108: 1-4Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), and aberrations thereof may underlie neuronal disorders. In view of the importance of the PSD in glutamatergic neurotransmission and its involvement in neuroplasticity, considerable efforts have been made to identify its protein constituents as a prelude to understand the molecular basis of PSD functioning. In the past several years, yeast two-hybrid technology has been extensively used to characterize proteins that interact with the glutamate receptors and may constitute core elements of the PSD involved in the regulation of receptor trafficking and signaling (reviewed in Refs. 9.Sheng M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7058-7061Crossref PubMed Scopus (290) Google Scholar, 10.McGee A.W. Bredt D.S. Curr. Opin. Neurobiol. 2003; 13: 111-118Crossref PubMed Scopus (84) Google Scholar, 11.Garner C.C. Nash J. Huganir R.L. Trends Cell Biol. 2000; 10: 274-280Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar). Based largely on these studies a protein-protein interaction map of the PSD has emerged. In brief, the model posits that the postsynaptic receptor complexes are localized by scaffolding proteins such as the synapse-associated protein (SAP) 90 (also known as PSD-95). These proteins link the receptors to various signaling molecules e.g. nitric-oxide synthase, calcium/calmodulin-dependent protein kinase II (CaMKII), and inositol 1,4,5-trisphosphate receptors. Receptors and scaffolding proteins are further linked to cytoskeletal proteins, the arrangement of which will determine the morphology of and protein trafficking in the spine. Yeast two-hybrid experiments are particularly useful in the detection of pairwise protein interactions, but are limited in their ability to reveal the global protein constituents of the protein complexes/organelles of interest. Alternatively, several PSD proteins have been identified from protein complexes by conventional biochemical means (12.Walsh M.J. Kuruc N. J. Neurochem. 1992; 59: 667-678Crossref PubMed Scopus (112) Google Scholar) or via the generation of antibodies against the protein complex for screening of the expression library (13.Langnaese K. Seidenbecher C. Wex H. Seidel B. Hartung K. Appeltauer U. Garner A. Voss B. Mueller B. Garner C.C. Gundelfinger E.D. Mol. Brain Res. 1996; 42: 118-122Crossref PubMed Scopus (63) Google Scholar). For a global analysis of proteins the recently developed mass spectrometric-based proteome approach is particular attractive because hundreds of proteins can be displayed and identified. The large dataset generated can be used to formulate hypotheses and in turn design experiments to understand the (distinct) physiological processes carried out by the differentially composed protein complexes. Recently, several large scale proteome and proteomics studies have been reported (4.Husi H. Ward M.A. Choudhary J.S. Blackstock W.P. Grant S.G. Nat. Neurosci. 2000; 3: 661-669Crossref PubMed Scopus (1038) Google Scholar, 5.Husi, H., Choudhary, J., Blackstock, W., and Grant, S. G. (2002) FENS Forum 2002 in Paris, Abstr. 112.11Google Scholar, 14.Bader G.D. Hogue C.W.V. Nat. Biotech. 2002; 20: 991-997Crossref PubMed Scopus (459) Google Scholar, 15.Koller A. Washburn M.P. Lange B.M. Andon N.L. Deciu C. Haynes P.A. Hays L. Schieltz D. Ulaszek R. Wei J. Wolters D. Yates J.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11969-11974Crossref PubMed Scopus (347) Google Scholar, 16.Cronshaw J.M. Krutchinsky A.N. Zhang W. Chait B.T. Matunis M.J. J. Cell Biol. 2002; 158: 915-927Crossref PubMed Scopus (784) Google Scholar, 17.Zhou Z. Licklider L.J. Gygi S.P. Reed R. Nature. 2002; 419: 182-185Crossref PubMed Scopus (734) Google Scholar, 18.Walikonis R.S. Jensen O.N. Mann M. William Provance D. Mercer J.A. Kennedy M.B. J. Neurosci. 2000; 20: 4069-4080Crossref PubMed Google Scholar). One of the first applications of proteome research in neuroscience was aimed at the characterization of novel PSD proteins (18.Walikonis R.S. Jensen O.N. Mann M. William Provance D. Mercer J.A. Kennedy M.B. J. Neurosci. 2000; 20: 4069-4080Crossref PubMed Google Scholar). The PSD fraction was isolated using a standard protocol developed by Carlin et al. (19.Carlin R.K. Grab D.J. Cohen R.S. Siekevitz P. J. Cell Biol. 1980; 86: 831-845Crossref PubMed Scopus (617) Google Scholar). The PSD proteins were partially separated on an SDS electrophoresis gel, trypsinized, and then characterized based on their peptide mass fingerprint (PMF). About thirty proteins, including "classic" and novel PSD proteins, were successfully elucidated. Overall, the number of proteins identified was lower than that expected, which may amount to above a hundred (4.Husi H. Ward M.A. Choudhary J.S. Blackstock W.P. Grant S.G. Nat. Neurosci. 2000; 3: 661-669Crossref PubMed Scopus (1038) Google Scholar, 9.Sheng M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7058-7061Crossref PubMed Scopus (290) Google Scholar, 10.McGee A.W. Bredt D.S. Curr. Opin. Neurobiol. 2003; 13: 111-118Crossref PubMed Scopus (84) Google Scholar). This suggested that a considerable number of PSD proteins remained to be characterized. In this study we aimed at a much higher coverage of the PSD protein content in order to reveal groups of proteins that would predict novel synaptic functions. We employed two methods for the separation and detection of the proteins, namely (a) high-load preparative two-dimensional gel electrophoresis to separate a larger number of PSD proteins with increased resolution, and identified the tryptic peptides of individual protein spots by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometer (MALDI TOF/TOF® MS), (b) isotope-coded affinity tagging (ICAT) of the proteins followed by trypsin digestion and the separation of cysteine-containing peptides by nano-liquid chromatography (LC) coupled online to an electrospray quadrupole-TOF mass spectrometer for the identification of the peptides (20.Tao W.A. Aebersold R. Curr. Opin. Biotech. 2003; 14: 110-118Crossref PubMed Scopus (247) Google Scholar). A large number of previously identified PSD proteins, as well as novel groups of proteins, were detected. Western blotting analysis of proteins from selected functional groups confirmed the enrichment of these proteins in the PSD. Grouping of the proteins based on their functions indicated protein complexes that are involved in diverse physiological activities, e.g. the receptors and ion channels, protein synthesis and breakdown, scaffolding, signal transduction etc. The wide diversity of proteins that may be active in the PSD clearly indicates the PSD to be the organizer of spine functioning sustaining the emerging view (21.Zhang W. Benson D.L. Hippocampus. 2000; 10: 512-526Crossref PubMed Scopus (54) Google Scholar, 22.Nimchinsky E.A. Sabatini B.L. Svoboda K. Annu. Rev. Physiol. 2002; 64: 313-353Crossref PubMed Scopus (940) Google Scholar) that considers the dendritic spine as the smallest self-sustaining (semi)-autonomous neuronal organelle. Purification of the PSD—The PSD fraction was isolated either as described (23.Wyneken U. Smalla K.-H. Marengo J.J. Soto D. de la Cerda A. Tischmeyer W. Grimm R. Boeckers T.M. Wolf G. Orrego F. Gundelfinger E.D. Neurosci. 2001; 102: 65-74Crossref PubMed Scopus (77) Google Scholar) based on the original method of Carlin et al. (19.Carlin R.K. Grab D.J. Cohen R.S. Siekevitz P. J. Cell Biol. 1980; 86: 831-845Crossref PubMed Scopus (617) Google Scholar), or based largely on a variant of the original method (24.Wu K. Carlin R. Siekevitz P. J. Neurochem. 1986; 46: 831-841Crossref PubMed Scopus (72) Google Scholar). In brief, in the original method, forebrains of 30 days old untreated rats were homogenized in homogenization buffer (5 mm Hepes, pH 7.4; 320 mm sucrose) containing a protease inhibitor mixture (Roche Applied Science). Cell debris and nuclei were removed by 1,000 × g centrifugation. The supernatant was spun for 20 min at 12,000 × g, resulting in supernatant and pellet P2. The P2 pellet was further fractionated by centrifugation in a sucrose step gradient to obtain the synaptosome, an organelle that contains both pre- and postsynaptic compartments. Synaptosome was lysed in hypotonic solution to release the cytoplasmic proteins and organelles such as mitochondria and small synaptic vesicle, and the resulting synaptic membrane was recovered by centrifugation using the sucrose gradient as stated above. For isolation of the PSD fraction, the synaptic membrane was diluted with 12 mm Tris-HCl (pH 8.1), 320 mm sucrose and an equal volume of 1% Triton X-100, 320 mm sucrose. The suspension was stirred for 15 min and then centrifuged for 30 min at 33,000 × g. The pellet was resuspended in 320 mm sucrose, 0.5% Triton X-100, 5 mm Tris/HCl, pH 8.1. After 15 min of stirring, the PSD proteins were pelleted by a 2-h centrifugation at 201,800 × g. All steps were carried out at 4 °C. This PSD preparation was used for all the two-dimensional gel experiments. In the variant method, the synaptic membrane was isolated as described above, and stirred for 30 min over ice in 1% Triton X-100 in 50 mm Hepes (pH 7.4). After centrifugation for 15 min at 30,000 × g, the pellet was suspended in 320 mm sucrose in Hepes buffer and loaded on top of a sucrose gradient consisting of 1 m, 1.5 m, and 2 m sucrose. The sample was centrifuged at 100,000 × g for 2 h, and the interface between 1.5 and 2 m was collected, mixed with equal volume of water containing 2% Triton X-100 and 150 mm KCl, and loaded directly on top of a sucrose gradient of 1.5/2 m. The gradient was centrifuged at 100,000 × g for 1 h. The interface at 1.5/2 m sucrose was collected, diluted 2× with water, and centrifuged to obtain the PSD fraction. After washing once with water, the pellet was redissolved in 0.5% SDS and then used for ICAT labeling, trypsin digestion, and liquid chromatography-tandem mass spectrometry studies. Two-dimensional Gel Electrophoresis—Two-dimensional gel electrophoresis was carried out as described (25.Jimenez C.R. Eyman M. Scotto Lavina Z. Gioio A. Li K.W. van der Schors R.C. Geraerts W.P.M. Giuditta A. Kaplan B.B. van Minnen J. J. Neurochem. 2002; 81: 735-744Crossref PubMed Scopus (56) Google Scholar). In brief, samples were solubilized in lysis buffer for 30 min (9 m urea, 2% CHAPS, 20 mm Tris, pH 7.5, 0.5% dithiothreitol, and 0.5% IPG buffer 3–10) and then centrifuged. 370 μl of the supernatant was used for the rehydration and simultaneously loading of the proteins to the IPG strip (Immobiline 18 cm DryStrip 3–10 NL, Amersham Biosciences), at 30 V for 12 h. The voltage was increased to 8000 V and focused for a total of 65,000 V/hr. Immediately after being focused, IPG strips were wrapped in plastic foil and stored at –80 °C. Prior to SDS-PAGE, IPG strips were equilibrated in 6 m urea/2% SDS/1% dithiothreitol/50 mm Tris, pH 8.8/30% glycerol for 15 min, followed by equilibration in 6 m urea/2% SDS/2.5% iodoacetamide/50 mm Tris, pH 8.8/30% glycerol for 15 min. The second dimension separation was run overnight using the Isodalt System (Amersham Biosciences) in 1.5-mm 11% gels (Duracryl, Genomic Solutions) at 25 mA per gel at 15 °C. After electrophoresis, gels were fixed and stained using either silver or colloidal Coomassie Brilliant Blue G-250. The gels were washed once with water and stored at room temperature in a plastic sealing until tryptic digestion. Digestion of Proteins from Two-dimensional Gels—All the visible protein spots from the Coomassie Blue-stained gel were manually excised with a round bottom dermal slicer of 3-mm diameter. The gel pieces were destained in 60% acetonitrile in 25 mm ammonium bicarbonate buffer, pH 8.5, and then dehydrated with 100% acetonitrile. The shrunken gel pieces were reswelled in 25 mm ammonium bicarbonate buffer, dehydrated again in 100% acetonitrile, and dried in a speedvac. For gel pieces that were heavily stained the rehydration/dehydration step was repeated once. The gel pieces were rehydrated in 8 μl of trypsin solution (20 μg/ml) for 1 h, followed by addition of 50 μlof25mm ammonium bicarbonate buffer to completely immerse the gel pieces. After incubation overnight at room temperature, 0.5 μl of the incubation buffer was pipetted to the MALDI plate and mixed with 1 μl of α-cyano-4-hydroxycinnamic acid. The samples were analyzed by a MALDI TOF/TOF® (Applied Biosystems) mass spectrometer (see below). In cases where the mass spectrometric signals were weak, the incubation buffer was loaded into a C18 Ziptip (Millipore) according to the protocol provided by the company. The bound peptides were eluted from the Ziptip using 1.0 μl of α-cyano-4-hydroxycinnamic acid, which was directly deposited onto the MALDI plate. The α-cyano-4-hydroxycinnamic acid matrix concentration was 5 mg/ml in 50% acetonitrile/50% water containing 0.1% trifluoroacetic acid. For the identification of (the abundant) proteins from the silver stained gel, protein spots were digested as stated above. After incubation with trypsin the supernatant was loaded into a self-packed Poros R2 (PerSeptive Biosystems) micro-tip, and eluted in 5 μl 50% acetronitrile/1% formic acid directly into a spraying electrode and analyzed by an electrospray Q-TOF (Micromass) mass spectrometer as described previously (26.Nagle G.T. de Jong-Brink M. Painter S.D. Li K.W. Eur. J. Biochem. 2001; 268: 1213-1221Crossref PubMed Scopus (26) Google Scholar) and below. Immunoblots—P2, synaptosome, synaptic membrane and PSD fraction were lysed in two-dimensional gel electrophoresis lysis buffer, except that the IPG buffer and bromphenol blue were omitted from the lysis buffer. Protein concentrations were determined by Bradford assay. Equal volume of the extract was mixed with 2× SDS buffer, boiled for 2 min, and 10 μg was loaded to a 10% mini SDS-gel. Electrophoresis was carried out at 120 V for 1 h, and the proteins were electrotransblotted to nitrocellulose membrane in the CAPSO buffer containing 10% methanol at 30V overnight. The membranes were blocked in 2% bovine serum albumin in Tris-buffered saline for 1 h, incubated in the primary antibodies (1:1000) for 1 h, washed in Tris-buffered saline containing 0.05% Tween 20, and incubated in the secondary antibodies (1:2000) for another hour. Signals were developed by enhanced chemiluminescence (Amersham Biosciences). The primary antibodies were purchased from Upstate Biotechnologies (EF-1α), BD Pharmingen (NMDA receptor subunit 1 clone 54.1), and Santa Cruz Biotechnology (sorting nexin 3). Anti PSD-95 was a gift from Dr. T. Südhof. The anti-ribosome serum was obtained from a patient with systemic Lupus erythematosus. All secondary antibodies were purchased from Dako. Mass Spectrometry of Trypsin-digested Protein Spots from Two-dimensional Gels—The mass spectrometer utilized for the high throughput protein analysis was an Applied Biosystems 4700 Proteomics Analyzer with TOF/TOF™ Optics. This MALDI tandem mass spectrometer uses a 200 Hz frequency-tripled neodinium YAG laser operating at a wavelength of 355 nm. For MS/MS, ions generated by the MALDI process were accelerated at 8 kV through a grid at 6.7 kV into a short, linear, field-free drift region. In this region the ions passed through a timed-ion selector (TIS) device that is able to select one peptide from a mixture of peptides at different m/z for subsequent fragmentation in the collision cell. After a peptide at a given m/z was selected by the TIS it passed through a retarding lens where the ions were decelerated and then passed into the collision cell, which was operated at 7 kV. The collision energy was defined by the potential difference between the source and the collision cell and hence was 1 kV. Inside the collision cell the selected peptide ions collided with air at a pressure of 1 × 10–6 Torr. After passing through the collision cell the ions (both intact peptide ion, the precursor, and fragments caused by collision with the air, the product ions) were reaccelerated in the second source region at 15 kV, passed through a second, field free, linear drift region, into the reflector and finally to the detector. The detector amplified and converted the signal to electrical current, which was observed and manipulated on a PC-based operating system. For reflector mode the operation of the instrument is far simpler. After the MALDI process generates the peptide ions they are accelerated at 20 kV through a grid at 14 kV into the first, short, linear, field-free drift region. After this point the rest of the instrument can be treated as a continuation of this field-free, drift region until the ions enter the reflector and then reach the detector where, as before, the signal at the detector is amplified and converted to electrical current. Both MS and MS/MS spectra were searched against the Mascot data base search engine (Matrix Science) to identify the proteins. Electrospray (tandem) mass spectrometric experiments were performed on a Micromass Q-TOF mass spectrometer as described previously (26.Nagle G.T. de Jong-Brink M. Painter S.D. Li K.W. Eur. J. Biochem. 2001; 268: 1213-1221Crossref PubMed Scopus (26) Google Scholar). The tryptic-digested samples were loaded into a nanoelectrospray capillary, which was pulled from a borosilicate glass capillary GC 100F-10 with a microcapillary puller. An internal wire electrode inserted inside the capillary was used for the measurement. The cone voltage was set at 25–30 V. For MS measurements, the quadrupole was operated in the rf-only mode and mass analysis was performed using the TOF analyzer. For tandem MS experiments, precursor ions were selected using the quadrupole, fragmented in the collision chamber using energies between 20 and 65 eV and argon as the collision gas, and the daughter ions detected by the TOF analyzer. Two major tryptic peptides were used for tandem MS, and the resulting daughter ion spectra were searched using the Mascot search engine. The electrospray MS that we used is less sensitive than the MALDI TOF/TOF® MS, and the potential co-migrating minor proteins in the protein spots are less likely to be detected. ICAT Labeling and Liquid Chromatography-Tandem Mass Spectrometry—The PSD fraction of about 0.1 mg was dissolved in 200 μl of 0.5% SDS in 50 mm Tris buffer, pH 7.5. It was then diluted 2 times in 50 mm Tris buffer, incubated with 1 mm TCEP, and labeled with ICAT® reagents according to the instruction of the ICAT labeling procedure as provided by the company (Applied Biosystems) with minor variations. Briefly, after neutralization of the ICAT® reagents, the samples were further diluted to a final concentration of 0.05% SDS. Trypsin at a ratio of 1:10 with respect to the sample protein concentration was added and incubated overnight at 37 °C. The digest was acidified and loaded into a4 × 15 mm cation exchange column equilibrated with 10 mm KH2PO4/25% acetonitrile, pH 3.0 at a flow rate of 1 ml/min. The column was washed with 3 column volumes of equilibration buffer to remove excess ICAT and other reagents and bound peptides were eluted from the column with 400 mm KCl in the same buffer and collected as single fraction. The fraction was neutralized and loaded into a 4 × 15 mm avidin column equilibrated in 2× phosphate-buffered saline. Non-ICAT-labeled peptides were removed by extensive washing with 2× phosphate-buffered saline, followed by washing with phosphate-buffered saline, 50 mm ammonium bicarbonate/20% methanol, pH 8.3 and water, and finally the bound ICAT-labeled peptides were eluted with 3 column volumes of 30% acetonitrile/0.4% trifluoroacetic acid. The eluted peptides were evaporated to dryness and reconstituted in 100 μl of cleavage reagent for 2 h at 37 °C to cleave the biotin portion of the tag from the labeled peptides. The fraction was dried, reconstituted in 150 μl of 5% acetonitrile/0.1% trifluoroacetic acid, and injected into a reversed phase capillary trap column at 40 μl/min. The peptides were resolved on a Dionex 75 μm × 15 cm C18 capillary column at 200 nl/min using a linear acetonitrile gradient from 5 to 50% in 60 min. The peptides were sprayed online into an electrospray quadrupole time-of-flight hybrid MS (QSTAR® from Applied Biosystems). Proteins were identified by analysis of collected MS/MS data using Pro ICAT™ software (Applied Biosystems). For two decades the 2× Triton extraction method to yield the PSD fraction from rat brain as developed by Carlin and coworkers (19.Carlin R.K. Grab D.J. Cohen R.S. Siekevitz P. J. Cell Biol. 1980; 86: 831-845Crossref PubMed Scopus (617) Google Scholar, 24.Wu K. Carlin R. Siekevitz P. J. Neurochem. 1986; 46: 831-841Crossref PubMed Scopus (72) Google Scholar) has been the standard protocol for the purification of PSD proteins and has been used in previous studies as the source for the characterization of novel PSD protein components. In the present study two independent proteome approaches were used, namely two-dimensional gel electrophoresis in conjunction with MALDI tandem mass spectrometry, and the ICAT peptide derivatization technique with nano-liquid chromatography coupled online to electrospray tandem mass spectrometry. In the first instance we used the two-dimensional gel-based method for the isolation of the PSD proteins. We verified the purification efficacy of the protocol resulting in the PSD fraction. We then completed the characterization of the proteins from the PSD fraction. First, we examined the enrichment of well-established PSD proteins in the PSD fraction, namely an NMDA receptor subunit NR1 and SAP90/PSD-95, and the depletion of a presynaptic marker protein, the integral synaptic vesicle membrane protein synaptophysin. Equal amounts of P2, synaptosome, synaptic membrane, and PSD fractions were run on an SDS gel, electroblotted onto nitrocellulose membranes and immunostained with antibodies against NR 1, SAP90/PSD-95, and synaptophysin, respectively. Fig. 1 reveals the high enrichment of the NR1 and SAP90/PSD-95 in the PSD fraction. Synaptophysin is enriched in the synaptosome, diminished in the synaptic membrane and totally absent from the PSD fraction. Second, the progress of purification was monitored by two-dimensional gel electrophoresis. 400-μg protein extracts of synaptosome, synaptic membrane, and the PSD fractions were separated by large format two-dimensional gels and stained with silver. The synaptosome fraction contains a high number of protein species over a wide range of isoelectric focusing point (pI) and Mr (Fig. 2A). The PSD fraction on the other hand exhibits a less complex protein pattern (Fig. 2C). Compared with the synaptic membrane a number of protein spots are relatively reduced in the PSD fraction, indicating that these proteins may not anchor to the PSD core (Fig. 2B). We then characterized nine of these proteins spots that are highly expressed in the synaptic membrane but apparently are absent or greatly reduced in the PSD fraction. Electrospray tandem mass spectrometry reveals that eight of them are mitochondrial proteins, namely aconitase, dihydrolipoamide succinyltransferase, ATP synthase α chain, H-ATP synthase subunit d, pyruvate dehydrogenase, isocitrate dehydrogenase, fumarase, and dihydrolipoamide acetyltranstransferase. Furthermore, there is a single glial cell-specific protein, the voltage-dependent anion-selective channel protein 1. Six of these proteins are not detectable in the PSD fraction (see Table I), and the other three proteins appear as minor spots. This shows that the Triton X-100 extraction step selectively removes proteins that are known to be exogenous to the PSD. But it also indicates that a small fraction of highly abundant proteins, notably mitochondrial and glial proteins, might survive the detergent extraction step and a minor fraction of them partitions into the PSD fraction.Table IMass spectrometric characterization of PSD proteins fractionated by two-dimensional gel electrophoresisSpotnrProtein nameNCBIDirect analysis mascot scoreZiptip mascot scoreMasspIPMFMS/MSPMFMS/MS1Peptidylprolyl isomerase A (cyclophilin A)839400986223181718.342Peptidylprolyl isomerase A (cyclophilin A)83940095767181718.343Peptidylprolyl isomerase A (cyclophilin A)839400974181718.344RIKEN cDNA 0810011J09 (similar to ubiquitin-conj-enzyme E2)2034185496165707.7456Sorting nexin 34507143118
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