Proteomic Analysis of in Vivo Phosphorylated Synaptic Proteins
2004; Elsevier BV; Volume: 280; Issue: 7 Linguagem: Inglês
10.1074/jbc.m411220200
ISSN1083-351X
AutoresMark O. Collins, Lu Yu, Marcelo P. Coba, Holger Husi, Iain Campuzano, Walter Blackstock, Jyoti S. Choudhary, Seth G. N. Grant,
Tópico(s)Retinal Development and Disorders
ResumoIn 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. 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. The molecular architecture of the synaptic junction has been studied intensely for many years, yielding information on its composition and function based on studies of individual receptors or small groups of proteins (1Sheng M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7058-7061Crossref PubMed Scopus (284) Google Scholar). In recent years, with the advent of proteomic technologies, a coherent map of the mammalian synapse proteome has been emerging. Mass spectrometry (MS) 1The abbreviations used are: MS, mass spectrometry; IMAC, immobilized metal affinity chromatography; IDA, iminodiacetic acid; TRA, targeted repeat analysis; NMDA, N-methyl-d-aspartate; NRC, NMDA receptor complex; IP3, inositol trisphosphate; PSD, postsynaptic density; LC, liquid chromatography; ESI, electrospray ionization; MOPS, 4-morpholinepropanesulfonic acid; PKA, protein kinase A; PKC, protein kinase C; SH3, Src homology 3.1The abbreviations used are: MS, mass spectrometry; IMAC, immobilized metal affinity chromatography; IDA, iminodiacetic acid; TRA, targeted repeat analysis; NMDA, N-methyl-d-aspartate; NRC, NMDA receptor complex; IP3, inositol trisphosphate; PSD, postsynaptic density; LC, liquid chromatography; ESI, electrospray ionization; MOPS, 4-morpholinepropanesulfonic acid; PKA, protein kinase A; PKC, protein kinase C; SH3, Src homology 3.-based analysis of the postsynaptic density (PSD) has established for the first time a detailed list of its molecular components (2Yoshimura Y. Yamauchi Y. Shinkawa T. Taoka M. Donai H. Takahashi N. Isobe T. Yamauchi T. J. Neurochem. 2004; 88: 759-768Crossref PubMed Scopus (178) Google Scholar, 3Peng J. Kim M.J. Cheng D. Duong D.M. Gygi S.P. Sheng M. J. Biol. Chem. 2004; 279: 21003-21011Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar, 4Li 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. J. Biol. Chem. 2004; 279: 987-1002Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Systematic analysis of functional multiprotein complexes embedded in the PSD (5Husi H. Ward M.A. Choudhary J.S. Blackstock W.P. Grant S.G. Nat. Neurosci. 2000; 3: 661-669Crossref PubMed Scopus (1012) Google Scholar) has also added to our knowledge of the overall organization of the postsynaptic proteome. Central to the functioning of signaling complexes and indeed the most basic signaling pathways is the process of reversible phosphorylation. The propagation of an appropriate synaptic response to receptor stimulation is highly regulated by phosphorylation cascades. This is exemplified by the process of synaptic plasticity, a process whereby glutamate receptor activation results in diverse signaling cascades, which ultimately lead to activation of transcription factors and modulation of gene expression. Phosphorylation is also employed to modulate protein function and stability and to mediate phosphorylation-dependent protein-protein interactions (e.g. Src homology 2 binding of phosphatidylinositol 3-kinase to NR2B) (6Hisatsune C. Umemori H. Mishina M. Yamamoto T. Genes Cells. 1999; 4: 657-666Crossref PubMed Scopus (77) Google Scholar), conferring a higher order level of regulation in such protein complexes. Historically, synapse phosphorylation and its importance in regulating neuronal signal transduction and brain function has been studied at the level of single molecules (7Greengard P. Valtorta F. Czernik A.J. Benfenati F. Science. 1993; 259: 780-785Crossref PubMed Scopus (1110) Google Scholar, 8Sweatt J.D. Kandel E.R. Nature. 1989; 339: 51-54Crossref PubMed Scopus (107) Google Scholar), but new proteomic strategies lend themselves to the global characterization of the signaling properties of the synapse proteome. Phosphopeptides can be purified from complex protein mixtures using immobilized metal affinity chromatography (IMAC) (9Andersson L. Porath J. Anal. Biochem. 1986; 154: 250-254Crossref PubMed Scopus (636) Google Scholar) and identified using MS (10Ficarro S.B. McCleland M.L. Stukenberg P.T. Burke D.J. Ross M.M. Shabanowitz J. Hunt D.F. White F.M. Nat. Biotechnol. 2002; 20: 301-305Crossref PubMed Scopus (1478) Google Scholar). Recent phosphoproteomic studies have utilized various peptide IMAC approaches, sometimes with methyl esterification, to enrich and improve specificity for phosphopeptides prior to MS. This approach has been successfully used to study phosphorylation in yeast (10Ficarro S.B. McCleland M.L. Stukenberg P.T. Burke D.J. Ross M.M. Shabanowitz J. Hunt D.F. White F.M. Nat. Biotechnol. 2002; 20: 301-305Crossref PubMed Scopus (1478) Google Scholar), Arabidopsis (11Nuhse T.S. Stensballe A. Jensen O.N. Peck S.C. Mol. Cell Proteomics. 2003; 11: 1234-1243Abstract Full Text Full Text PDF Scopus (518) Google Scholar), and cell lines (12Shu H. Chen S. Bi Q. Mumby M. Brekken D.L. Mol. Cell Proteomics. 2004; 3: 279-286Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). However, the application of these approaches to complex mammalian subcellular organelles such as the synapse has yet to be established. MS, although a powerful tool for analysis of protein phosphorylation, has several on-going technical challenges. In particular, these result from heterogeneity arising from dynamic site occupancy, multiple phosphorylation sites in the same low abundance peptide, and inherently poor fragmentation of phosphopeptides. Continued improvements in three critical areas, unbiased sample enrichment methods, high sensitivity MS, and data analysis methods, are required to fully harness the potential of MS. The field is in a phase of rapid development, and various strategies for protein or modification centric analyzes are being explored (13Beausoleil S.A. Jedrychowski M. Schwartz D. Elias J.E. Villen J. Li J. Cohn M.A. Cantley L.C. Gygi S.P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12130-12135Crossref PubMed Scopus (1227) Google Scholar, 14Chang E.J. Archambault V. McLachlin D.T. Krutchinsky A.N. Chait B.T. Anal. Chem. 2004; 76: 4472-4483Crossref PubMed Scopus (73) Google Scholar). Here, we describe the use of a combination of cellular fractionation procedures with large scale IMAC phosphoprotein and phosphopeptide enrichment protocols and complementary MS analytical strategies to characterize mouse forebrain synaptosomes. This has resulted in the unambiguous identification of 289 sites of phosphorylation in 79 synaptic proteins involved in important pre- and postsynaptic multiprotein complexes and signaling pathways. Large scale in vitro phosphorylation screening on peptide arrays for 95 sites with seven kinases identified 28 phosphorylated sites and a total of 52 phosphorylation events. The simultaneous identification of large numbers of sites of phosphorylation and identification of responsible kinases, as exemplified by this study, is a powerful approach to expand the current knowledge of cell signaling in a particular system. Isolation of Synaptosomes—Synaptosomes were prepared as described by Carlin et al. (1980) (15Carlin R.K. Grab D.J. Cohen R.S. Siekevitz P. J. Cell Biol. 1980; 86: 831-845Crossref PubMed Scopus (597) Google Scholar) with minor modifications. In brief, mouse forebrains were homogenized in a cold buffer containing 50 mm Tris acetate pH 7.4, 10% (w/w) sucrose, 5 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 2 mg/ml aprotinin, and 2 mg/ml leupeptin. The sample was then centrifuged for 20 min at 800 × g, and the resulting supernatant was centrifuged again for 30 min at 16,000 × g. The pellet was then resuspended in 5 ml/g of original weight in a buffer containing 5 mm Tris acetate, pH 8.1, 1 mm phenylmethylsulfonyl fluoride, 2 mg/ml aprotinin, and 2 mg/ml leupeptin, quickly homogenized, and incubated for 45 min on ice. After a further homogenization step and the addition of sucrose to 34% (w/w), the sample was overlaid with solutions containing 28.5% (w/w) sucrose in 50 mm Tris acetate, pH 7.4, and 10% (w/w) sucrose in 50 mm Tris acetate, pH 7.4, and centrifuged for 2 h at 60,000 × g at 4 °C. The protein-containing band, which formed between the 34 and 28.5% sucrose gradients, was collected and diluted with 50 mm Tris acetate, pH 7.4, to 10% sucrose and then centrifuged for 30 min at 48,000 × g. The pellet was then resuspended in 50 mm Tris acetate, pH 7.4, and homogenized gently to form the synaptosomal preparation. Protein IMAC of Urea-soluble Synaptosomal Fraction—Fast-flow chelating Sepharose with iminodiacetic acid (IDA) (Amersham Biosciences) or nitrilotriacetic acid (Qiagen) chelating groups were charged with GaCl3 or FeCl3. Synaptosomal proteins (12.5 mg) were solubilized in 6 m urea, and the supernatant was removed and incubated with 2 ml of the metal charged resin with mixing for 1 h at room temperature. The unbound protein was washed with buffer A (6 m urea, 50 mm Tris acetate) to base line, and the phosphoproteins were specifically eluted with buffer B (6 m urea, 50 mm Tris acetate, 100 mm EDTA, 100 mm EGTA). The fractions were collected, concentrated, and washed with buffer B in a Vivaspin 6 PES membrane spin column (Vivascience). 13.4 μg of protein was diluted with buffer C (1 m urea, 0.125 m thiourea, 5% (v/v) CH3CN, and 0.1 m NH4HCO3) to a final volume of 400 μl. Trypsin (sequence grade; Roche Applied Science) was added to the sample in a ratio of 1:20, and the mixture was incubated at 37 °C for 2 h and then dried in a SpeedVac (Thermo Life Science). The sample was reconstituted in 50 mm NH4HCO3, and one-quarter was injected for each on-line LC-MS/MS analysis. Double IMAC of Urea-soluble Synaptosomal Fraction—3 mg of protein IMAC purified sample was digested with modified porcine or gold trypsin (Promega) in a ratio of 1:20 in buffer D (1 m urea and 25 mm NH4HCO3) at 37 °C for 4 h. The resultant digest was desalted and dried, and methyl esterification was performed with 2 m methanolic HCl (10Ficarro S.B. McCleland M.L. Stukenberg P.T. Burke D.J. Ross M.M. Shabanowitz J. Hunt D.F. White F.M. Nat. Biotechnol. 2002; 20: 301-305Crossref PubMed Scopus (1478) Google Scholar) when needed. Self Pack POROS® 20 MC media (Applied Biosystems) for phosphopeptide purification was charged with GaCl3 as described above for the IDA/nitrilotriacetic acid resins. Peptide digests (with and without methyl esterification) were reconstituted in buffer E (equal volumes of acetonitrile, methanol, and water, pH 2.5-3). 1 ml of this peptide mixture was incubated with 200 μl of POROS-Ga slurry for 1 h at room temperature. The resin was then loaded into a spin column and washed with 10 volumes of buffer E. Phosphopeptides were eluted with 2 × 100 μl of 200 mm Na2HPO4. Peptide IMAC of Whole and Urea-insoluble Synaptosomal Fractions—2.5 mg of the 6 m urea insoluble pellet left after removal of the supernatant was digested with trypsin in buffer F (2 m urea and 25 mm NH4HCO3). The resultant digest was desalted, methyl-esterified, and subjected to a peptide IMAC step as described previously. Similarly, 6 mg of whole synaptosomal proteins were digested in buffer F, desalted, methyl-esterified, and subjected to a peptide IMAC step. Phosphoprotein Staining and Image Analysis—IMAC-enriched phosphoprotein samples were separated on a 12% SDS-polyacrylamide gel and sequentially stained with Pro-Q diamond (phosphoprotein) and SYPRO Ruby (total protein) stains (Molecular Probes, Inc., Eugene, OR). Peppermint Stick phosphoprotein molecular weight standards (Molecular Probes) served as a molecular weight marker and internal control. Images were captured with a Typhoon scanner (Amersham Biosciences) and overlaid using TotaLab software (Nonlinear Dynamics). On-line Nano-LC-MS/MS—A nanoflow high pressure liquid chromatography system, Ultimate™ (LC Packings) or CapLC (Waters), was coupled to a Q-ToF 1, Q-ToF Ultima (Waters/Micromass), or 4000 QTRAP (Applied Biosystems). Tryptic peptides from the phosphoprotein digest were loaded in 0.1% aqueous formic acid and desalted on PepMap C18 trapping cartridge (LC Packings). BetaMax Neutral (Thermo Hypersil-Keystone) was used to trap the IMAC-enriched phosphopeptides in 0.5% aqueous formic acid. Peptides on the trap were back-flushed to and separated on the analytical column (PepMap C18, 75-μm inner diameter × 15 cm; LC Packings). The gradients are shown in Tables I and II.Table ILC gradient for separation of phosphoprotein digest Solvent A was 95% H2O, 5% ACN, 0.1% formic acid; solvent B was 95% ACN, 5% H2O, 0.1% formic acid. Flow rate through the columns in the separation was 200-250 nl/min.Solvent0 min180 min240 min242 min247 minA (%)9430502020B (%)670508080 Open table in a new tab Table IILC gradient for IMAC-enriched phosphopeptide analysis Solvent A was 100% H2O, 0.1% formic acid; solvent B was 70% ACN, 30% H2O, 0.1% formic acid. Flow rate through the columns in the separation was 150 nl/min.Solvent03075105125135145150160A (%)9998979590805055B (%)12351020509595 Open table in a new tab In the LC-MS/MS analysis of the phosphoprotein digest, the Q-ToF Ultima was operated in automated data-dependent acquisition mode. Each cycle had a 1-s MS survey (m/z 400-1500), and up to three of the highest intensity multiply charged ions (+2 and +3) were selected for MS/MS (m/z 50-2000), each for 5 s. The collision energy in MS/MS was varied according to the m/z and the charge state of the precursor ion. Due to the high complexity of the sample, there were two LC-MS/MS runs with the same LC gradient. After the first standard run (Run 1), survey data were examined, and multiply charged ions above the intensity threshold that had not been subjected to MS/MS in Run 1 were incorporated into the inclusion list for the second run (Run 2). We term this latter LC-MS/MS approach, incorporating a primary analysis followed by a repeat experiment based on an inclusion list, targeted repeat analysis (TRA). Analysis of IMAC-enriched phosphopeptides was performed on the Q-ToF using similar acquisition parameters to those for the Q-ToF Ultima. Two different precursor-scanning approaches were adopted on the 4000 QTRAP to selectively analyze phosphopeptides. In the first approach, the instrument was used in negative ion mode (-ESI) to scan for the precursors of m/z 79 (PO3−) with automatic switching to positive mode (+ESI) for MS/MS for the detected precursors. In the second approach, we applied TRA strategy. First, one-third of the sample was analyzed in negative mode for detecting precursors of m/z 79. Data were examined to generate the inclusion list for the second run in positive ion mode (+ESI) using the remaining two-thirds of the sample in information-dependent acquisition mode. Raw data were processed to give a peak list file and submitted to a local Mascot version 2.0 (Matrix Science) server for iterative searching on a custom, nonidentical, combined human and mouse IPI data base (EBI). Assignment of phosphorylation sites was verified manually with the aid of PEAK Studio (Bioinformatics Solutions) software. Peptide Array Phosphorylation Assays—Jerini Phosphosite detector™ peptide arrays (Jerini Peptide Technologies, Gmbh) were used to determine which of the MS-identified phosphorylation sites (95 selected sites) could be phosphorylated by seven kinases in an in vitro assay. 15-amino acid-long peptides that encompassed the selected sites were synthesized on cellulose membranes in a parallel manner using SPOT technology (16Wenschuh H. Volkmer-Engert R. Schmidt M. Schulz M. Schneider-Mergener J. Reineke U. Biopolymers. 2000; 55: 188-206Crossref PubMed Scopus (173) Google Scholar) deposited to glass slides and were covalently immobilized to the glass slide surface. Each peptide was present in triplicate on the chip, and seven full-length proteins that are capable of being phosphorylated were also included. Negative control peptides for each phosphorylation site were included, replacing serine or threonine with alanine or valine, respectively, and positive control peptide sequences for each kinase were present. Peptide arrays were sealed with Gene-Frame™ incubation chambers (Abgene house, Surrey, UK), and the chambers were filled with 330 μl of kinase buffer (20 mm MOPS, pH 7.2, 25 mm β-glycerol phosphate, 5 mm, EGTA, 1 mm sodium orthovanadate, 1 mm dithiothreitol, 100 μm ATP, 15 mm MgCl2, and 10 μm [γ-32P]ATP). Recombinant active kinases (Upstate Biotechnology, Inc., Lake Placid, NY) (3 μg of PKA catalytic subunit (with 2 μm cAMP), 2 μg of Akt1 (Δ PH, S473D), 2 μg of Erk1, 3 μg of p38α, 3 μg of CKII, 3 μg of Cdk5/p35, and 1.5 μg of PKC (α, β, γ)) were included in the appropriate kinase assays. In the case of PKC, a modified kinase buffer was used (10 mm MOPS, pH 7.2, 12.5 mm β-glycerol phosphate, 2.5 mm EGTA, 0.5 mm sodium orthovanadate, 0.5 mm dithiothreitol, 0.5 mm CaCl2, 0.1 mg/ml phosphatidylserine, 25 μg/ml diacylglycerol). After a 45-min incubation at 32 °C, the peptide microarrays were washed six times, alternating between 0.1 m phosphoric acid and distilled water. γ-32P incorporation in the immobilized peptide spots was detected on a Typhoon 8600 PhosphorImager (50-μm resolution; Amersham Biosciences). Image analysis and signal quantification was carried out using ImageQuant TL (Amersham Biosciences), and positive signals were defined after background subtraction. Bioinformatic Analysis—All proteins detected in this study were classified according to Swiss-Prot keywords (available on the World Wide Web at ca.expasy.org/sprot/). Known phosphoproteins in Supplementary Table I were annotated by extensive literature mining of PubMed. Scansite (available on the World Wide Web at scansite.mit.edu/) was used in high stringency mode to predict if proteins in Supplementary Table I were likely to be phosphorylated, and responsible kinases are indicated. TMHMM Server version 2.0 (available on the World Wide Web at www.cbs.dtu.dk/services/TMHMM/) was used to predict the occurrence of transmembrane helices. Scansite and NetPhosK (available on the World Wide Web at www.cbs.dtu.dk/services/NetPhos/) were both used for predicting phosphorylation sites by cognate kinases and on those peptides where the phosphorylation sites could not be unambiguously assigned by MS data (Supplemental Table III). Scansite was also used to check the presence of phosphorylation sites in Pfam protein domains and to predict whether phosphorylation sites were localized in phosphodependent interaction domains (Supplemental Table III). Graphical illustrations of the location of identified sites in relation to protein domain structure, three-dimensional structure, and information on homologs and paralogs of identified phosphorylation sites can be found on the World Wide Web at www.ppo4.org/phospho/synaptosome.html. Network Construction—Protein-protein interactions for the N-methyl-d-aspartate (NMDA) receptor complex proteins were obtained from PPID (available on the World Wide Web at www.PPID.org). Each protein (node) that had interaction (edge) information was used to plot a network graph using InterViewer, a network graphing program. Kinase-substrate edges were manually superimposed onto the protein-protein interaction network. Several approaches capable of identifying large numbers of in vivo phosphorylated proteins have been used to functionally annotate the mouse synaptic proteome. A summary of this is shown in Fig. 1A. In the first approach, a mouse synaptosomal preparation was digested with trypsin and phosphopeptides captured by peptide IMAC. The enriched sample was analyzed by nano-flow LC-MS/MS. Despite the enrichment, the majority of identifications corresponded to unphosphorylated peptides. In the second approach, the synaptosomes were first divided into urea soluble and insoluble fractions, the latter containing mostly membrane-bound components. Peptide IMAC and MS was performed on the insoluble fraction in a comparable manner to the first approach. For the urea-soluble fraction, a new strategy was adopted, entailing an additional stage of enrichment by capture of the phosphoproteins. A two-pronged route was taken for subsequent analysis of the trypsin digest of this enriched fraction. The first route is based on rigorous MS using (a) LC-MS/MS peptide identification based on a TRA approach described under "Experimental Procedures" and (b) precursor ion scanning to identify phosphopeptides prior to sequencing. The second route used a further peptide IMAC followed by LC-MS/MS. Using these combined approaches and implementing a previously unreported strategy of sequential protein and peptide IMAC, we have identified 228 potential synaptic phosphoproteins (Supplementary Table I) and characterized over 350 phosphopeptides containing 331 sites of phosphorylation (Supplemental Tables II and III). Protein IMAC Protocol—Unlike peptide IMAC that is widely used for phosphoproteomic analysis, to our knowledge protein IMAC approaches have not been reported. In order to develop a protein IMAC protocol, we tested two resins: Sepharose-IDA (a tridentate ligand) and agarose-nitrilotriacetic acid (a quadradentate ligand) and two metal ions, FeCl3 and GaCl3 (Fig. 1B), to isolate phosphoproteins from urea-soluble preparations. The IDA resin with both metal ions showed marked enrichment indicated by specific phosphoprotein staining on SDS-polyacrylamide gels. The Ga3+ resin showed more effective depletion of phosphoprotein from the unbound fraction together with stronger phosphostaining of the purified sample (Fig. 1C) and therefore was used in all subsequent protein IMAC experiments. Analysis of tryptic peptides from the protein IMAC enrichment by LC-MS/MS identified 152 proteins and 19 phosphopeptides (Supplementary Tables I and II). TRA extended this by an additional 30 protein identifications and 21 phosphopeptides. A further 28 different phosphopeptides from these samples were identified using the precursor-scanning approach, applied in the routine as well as the targeted manner as described under "Experimental Procedures." Clearly, the TRA strategy is effective in both standard LC-MS/MS and precursor scanning modes for extending both protein and phosphorylation mapping from complex peptide mixtures. 186 proteins have been identified in the protein IMAC sample. 105 of these have been confirmed as phosphoproteins in the literature and by phosphorylation sites identified in this study (Fig. 2). This suggests that whereas a small proportion may represent contaminating proteins, the majority are probably phosphoproteins. The identified components represent a diverse range of protein classes (Fig. 2B and Supplemental Table I), and low abundance protein classes, such as kinases, phosphatases, and small G-proteins and modulators, are well represented even in the presence of very abundant cytoskeletal proteins. The protein IMAC approach presents important benefits for phosphorylation analysis. It offers the opportunity to use basic protein identification to identify many candidate phosphoproteins, which are of low abundance and would not have been in the dynamic range required for direct phosphorylation analysis. The majority of phosphopeptides characterized in MS analyses of the phosphoprotein mixture cover single phosphorylation events (Fig. 3) and thus are complementary to measurements made from the peptide IMAC approach that better represents highly phosphorylated peptides. A further important benefit is that it provides scope for two stages of sample enrichment, at the protein level and subsequently at the peptide level. Double IMAC Protocol—LC-MS/MS analysis of phosphopeptide from double IMAC yielded identification and characterization of 176 phosphopeptides (Fig. 2A and Supplemental Table II) derived from 41 phosphoproteins. The results from the double IMAC protocol significantly extended those from the protein IMAC protocol on several levels. First, of the 25 validated phosphoproteins found in the protein IMAC experiments, we found an additional 115 phosphopeptides on 16 proteins, indicating greater depth of analysis. We also identified a further 14 phosphopeptides corresponding to seven proteins detected in the protein IMAC sample. Second, 19 phosphoproteins containing 37 phosphopeptides that were not characterized in the protein IMAC were observed in the double IMAC protocol. Third, the increased depth of phosphorylation coverage through overlapping peptides reflects variability in site occupation that can occur on a protein and a level of heterogeneity associated with this type of modification. This is well illustrated in Bassoon, for which we found 15 phosphorylation sites (10 peptides) in the protein IMAC protocol and 16 new sites (16 peptides) in the double IMAC protocol (Supplemental Table III). It is evident that combination of protein and double IMAC strategies enables identification and characterization of phosphoproteins across a wider range of protein abundance and phosphorylation states. Synaptic Membrane Phosphoproteomic Analysis—Integral membrane proteins are usually difficult to analyze, since detergent-based extraction methods are not readily compatible with subsequent purification strategies and LC-MS/MS analysis. However, for a phosphoproteomic analysis, cytoplasmic domains of integral membrane proteins are sufficient, since they contain the phosphorylation sites involved in intracellular signaling. Therefore, we digested 6 m urea-insoluble fractions of synaptosomes; the supernatant was desalted and esterified as described above and was subjected to a single peptide IMAC and LC-MS/MS analysis (Fig. 1A). This approach allowed identification and characterization of 60 phosphopeptides from 31 proteins (Supplemental Table II), of which 12 are predicted to be integral membrane proteins and many others, such as PSD-95 and Adapter-related protein complex-2 α-1, are known to be membrane-associated. 10 of the identified membrane proteins were not previously known to be phosphoproteins. Of these 31 phosphoproteins, 18 were not detected in either protein or double IMAC protocols (Fig. 3A). Complementarity of Analytical Approaches—Inspection of the summarized data (Figs. 2A and 3) shows that the combination of subcellular fractionation and IMAC protocols with MS analyses are complementary, both in terms of the protein identification and phosphorylation site characterization. Overall, we sequenced 350 phosphopeptides covering 653 phosphorylation events. These correspond to a total of 331 phosphorylation sites, of which 289 were localized unambiguously (Fig. 1A). The phosphorylation sit
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