Quantitative Analysis of Synaptic Phosphorylation and Protein Expression

Abstract

The postsynaptic density (PSD) signaling machinery contains proteins with diverse functions. Brain region-specific variations in PSD components mediate distinct physiological responses to synaptic activation. We have developed mass spectrometry-based methods to comprehensively compare both relative protein expression and phosphorylation status from proteins present in biochemical preparations of postsynaptic density. Using these methods, we determined the relative expression of 2159 proteins and 1564 phosphorylation sites in PSD preparations from murine cortex, midbrain, cerebellum, and hippocampus. These experiments were conducted twice using independent biological replicates, which allowed us to assess the experimental and biological variability in this system. Concerning protein expression, cluster analysis revealed that known functionally associated proteins display coordinated synaptic expression. Therefore, proteins identified as co-clustering with known protein complexes are prime candidates for assignment as previously unrecognized components. Concerning degree of phosphorylation, we observed more extensive phosphorylation sites on N-methyl-d-aspartate (NMDA) receptors than α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, consistent with the central role of N-methyl-d-aspartate receptors in processing synaptic transmission patterns. Average kinase and phosphatase levels were highest in the hippocampus, correlating with a higher overall phosphopeptide abundance present in this brain region. These findings suggest that the hippocampus utilizes reversible protein phosphorylation to a greater extent than other brain regions when modifying synaptic strength. The postsynaptic density (PSD) signaling machinery contains proteins with diverse functions. Brain region-specific variations in PSD components mediate distinct physiological responses to synaptic activation. We have developed mass spectrometry-based methods to comprehensively compare both relative protein expression and phosphorylation status from proteins present in biochemical preparations of postsynaptic density. Using these methods, we determined the relative expression of 2159 proteins and 1564 phosphorylation sites in PSD preparations from murine cortex, midbrain, cerebellum, and hippocampus. These experiments were conducted twice using independent biological replicates, which allowed us to assess the experimental and biological variability in this system. Concerning protein expression, cluster analysis revealed that known functionally associated proteins display coordinated synaptic expression. Therefore, proteins identified as co-clustering with known protein complexes are prime candidates for assignment as previously unrecognized components. Concerning degree of phosphorylation, we observed more extensive phosphorylation sites on N-methyl-d-aspartate (NMDA) receptors than α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, consistent with the central role of N-methyl-d-aspartate receptors in processing synaptic transmission patterns. Average kinase and phosphatase levels were highest in the hippocampus, correlating with a higher overall phosphopeptide abundance present in this brain region. These findings suggest that the hippocampus utilizes reversible protein phosphorylation to a greater extent than other brain regions when modifying synaptic strength. Synaptic transmission between neurons in the central nervous system involves the release of neurotransmitter from presynaptic neurons and its detection by specific ligand-gated ion channels in the surface membrane of postsynaptic neurons. These neurotransmitter receptors exist as part of a highly organized protein complex known as the postsynaptic density (PSD). 1The abbreviations used are: PSD, postsynaptic density; CaMKII, calcium/calmodulin-dependent protein kinase II; GluR, ionotropic glutamate receptor; NMDA, N-methyl-d-aspartate; SCX, strong cation exchange; SOM, self-organizing map; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; TARP, transmembrane AMPA receptor regulatory protein; iTRAQ, isobaric tagging for relative and absolute quantitation; MINT, Molecular INTeraction; Qq-Tof, quadrupole selecting, quadrupole collision cell, time-of-flight. 1The abbreviations used are: PSD, postsynaptic density; CaMKII, calcium/calmodulin-dependent protein kinase II; GluR, ionotropic glutamate receptor; NMDA, N-methyl-d-aspartate; SCX, strong cation exchange; SOM, self-organizing map; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; TARP, transmembrane AMPA receptor regulatory protein; iTRAQ, isobaric tagging for relative and absolute quantitation; MINT, Molecular INTeraction; Qq-Tof, quadrupole selecting, quadrupole collision cell, time-of-flight. In addition to neurotransmitter receptors, the PSD is composed of proteins of diverse function (1Ziff E.B. Enlightening the postsynaptic density.Neuron. 1997; 19: 1163-1174Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 2Kennedy M.B. Signal-processing machines at the postsynaptic density.Science. 2000; 290: 750-754Crossref PubMed Scopus (657) Google Scholar). These functional groups include scaffolding molecules, kinases and phosphatases, G proteins and their effectors, and adhesion proteins. The coordinated functioning of the different PSD components regulates in part the strength of signaling between the pre- and postsynaptic neurons. At the molecular level, this regulation can be achieved by alterations in protein localization (3Malenka R.C. Synaptic plasticity and AMPA receptor trafficking.Ann. N. Y. Acad. Sci. 2003; 1003: 1-11Crossref PubMed Scopus (268) Google Scholar, 4Colbran R.J. Brown A.M. Calcium/calmodulin-dependent protein kinase II and synaptic plasticity.Curr. Opin. Neurobiol. 2004; 14: 318-327Crossref PubMed Scopus (255) Google Scholar, 5Smith K.E. Gibson E.S. Dell'Acqua M.L. cAMP-dependent protein kinase postsynaptic localization regulated by NMDA receptor activation through translocation of an protein kinase A anchoring protein scaffold protein.J. Neurosci. 2006; 26: 2391-2402Crossref PubMed Scopus (123) Google Scholar), reversible post-translational modifications (e.g. phosphorylation (6Soderling T.R. Derkach V.A. Postsynaptic protein phosphorylation and LTP.Trends Neurosci. 2000; 23: 75-80Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar, 7Yamauchi T. Molecular constituents and phosphorylation-dependent regulation of the post-synaptic density.Mass Spectrom. Rev. 2002; 21: 266-286Crossref PubMed Scopus (62) Google Scholar) and O-GlcNAc glycosylation (8Vosseller K. Trinidad J.C. Chalkley R.J. Specht C.G. Thalhammer A. Lynn A.J. Snedecor J.O. Guan S. Medzihradszky K.F. Maltby D.A. Schoepfer R. Burlingame A.L. O-Linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry.Mol. Cell. Proteomics. 2006; 5: 923-934Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar)), and changes in protein levels via local synthesis and degradation (9Ehlers M.D. Activity level controls postsynaptic composition and signaling via the ubiquitin-proteasome system.Nat. Neurosci. 2003; 6: 231-242Crossref PubMed Scopus (823) Google Scholar). Integration of these processes may be the basis of long term changes in synaptic efficacy thought to underlie higher cognitive processes such as learning and memory (10Bliss T.V. Collingridge G.L. Morris R.G. Introduction. Long-term potentiation and structure of the issue.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2003; 358: 607-611Crossref PubMed Scopus (97) Google Scholar). Proteomics approaches aimed at identifying synaptic proteins (11Li 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, 12Peng 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, 13Yoshimura Y. Yamauchi Y. Shinkawa T. Taoka M. Donai H. Takahashi N. Isobe T. Yamauchi T. Molecular constituents of the postsynaptic density revealed by proteomic analysis using mass spectrometry.J. 2004; PubMed Scopus Google Scholar, M. E.B. and of postsynaptic density Cell. Proteomics. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, J.C. Specht C.G. Thalhammer A. Schoepfer R. Burlingame A.L. of phosphorylation sites in postsynaptic density Cell. Proteomics. 2006; 5: Full Text Full Text PDF PubMed Scopus Google Scholar, J.C. Thalhammer A. Specht C.G. Schoepfer R. Burlingame A.L. of postsynaptic density PubMed Scopus Google Scholar) and their post-translational modifications (8Vosseller K. Trinidad J.C. Chalkley R.J. Specht C.G. Thalhammer A. Lynn A.J. Snedecor J.O. Guan S. Medzihradszky K.F. Maltby D.A. Schoepfer R. Burlingame A.L. O-Linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry.Mol. Cell. Proteomics. 2006; 5: 923-934Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, M.P. H. analysis of in synaptic Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. C.G. A. analysis of from PubMed Scopus Google Scholar) have of synaptic is to biological function to sites on their in a proteomics the role of specific proteins and phosphorylation is to their synaptic levels with changes of physiological and as as in the central nervous system. the relative expression of synaptic proteins in brain and absolute conducted on a of proteins D. J. Duong D.M. J. T. Sheng M. J. and absolute of postsynaptic density from rat forebrain and Cell. Proteomics. 2006; 5: Full Text Full Text PDF PubMed Scopus (382) Google Scholar). A phosphorylation in and on changes in a of sites R. M. M. D. D. B. R. and of protein phosphorylation in and Cell. Proteomics. 6: Full Text Full Text PDF PubMed Scopus Google Scholar). using mass can be using methods M. M. M. R. mass and for the analysis of protein PubMed Scopus Google Scholar). In the present we PSD complexes from the cortex, midbrain, cerebellum, and hippocampus and used the with B. K. S. N. S. S. S. S. S. M. A. protein in using isobaric tagging Cell. Proteomics. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) to protein abundance on 2159 proteins and 1564 sites of We that a of highly expression that these highly correlating protein are candidates for We in the of sites at levels of phosphorylation of NMDA receptors relative to AMPA we that proteins in kinase signaling have a higher overall abundance in the hippocampus and that their phosphorylation is higher than in other brain The conducted twice using of PSD that were at as previously J.C. Specht C.G. Thalhammer A. Schoepfer R. Burlingame A.L. of phosphorylation sites in postsynaptic density Cell. Proteomics. 2006; 5: Full Text Full Text PDF PubMed Scopus Google Scholar). from of were the by the The brain regions were to as The from the The of the Brain regions cortex, hippocampus, and were in and from to the biochemical The brain in a a of phosphatase and by brain the of to brain of to that PSD to an level of phosphatase The on a density and by Synaptic were at the and a The PSD at the and Average of PSD brain for regions were as for cortex, for for cerebellum, and for hippocampus. of PSD were in PSD in The for at with to these were with in the for at The with and The to and the for at The were using a and to using a The were in of using of and a of were used to of the of with a specific brain PSD as the with the regions with the with and the hippocampus with In the of the the of the brain regions The allowed to for at using a and as a to that of identified A a of the by to for protein to be the of the chromatography using an with a in with A The PSD the in B of A with The from B to B from B to B and from B to B and fractions were and using a and using a of for analysis using and the to conducted on the of using an were using M.J. B. A.J. at the level of from using and Chem. 2004; PubMed Scopus Google Scholar, of from using Cell. Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar) an with a from fractions were in of and the with an of to by of were from the a using of The with of were from the using of and this to using a and fractions were using a at a of a in acid on an system with an were between and in on The to a to a mass were in ion were for the were for of ion the quadrupole to which for transmission of of the The and A that the from for its were using and were using the The were the with a of using the in and were and as the and to were and of were as The were as protein of allowed as a of were allowed for from were used to ion that were with a in of The of both a for and proteins were identified were identified with at with a and a The were by the using a the as as a of the were the and proteins were to proteins for and to the that to proteins from more than were used for in of using with an overall protein of and a of to a of with a of were to that the of abundance were when were with the of an assignment The in were using the of the at and were using by the for these of the were for the at the the used for the relative of a in of the the of that by the for in that protein expression for protein were the of to that phosphorylation were by relative phosphorylation levels by relative protein the were used were Using the M. T. J. J. 2006; PubMed Scopus Google Scholar), protein expression were both biological to self-organizing of protein expression to cluster both proteins as as brain regions using the M.B. Brown D. analysis and display of expression Acad. Sci. S. A. PubMed Scopus Google Scholar). The relative expression were and by brain and and by both the and used as the the in of their to cluster we known as in the Molecular A. M. M. a Molecular 2002; PubMed Scopus Google Scholar). known between proteins A of protein in the for which both were in of The determined for of these proteins. The were and other analysis conducted using the on the the of were the used to specific compare the relative of protein and phosphorylation brain we PSD from of cerebellum, cortex, hippocampus, and PSD with and the were with of the of the to for of relative abundance levels B. K. S. N. S. S. S. S. S. M. A. protein in using isobaric tagging Cell. Proteomics. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). were and a used to and proteins and phosphorylation sites as previously J.C. Specht C.G. Thalhammer A. Schoepfer R. Burlingame A.L. of phosphorylation sites in postsynaptic density Cell. Proteomics. 2006; 5: Full Text Full Text PDF PubMed Scopus Google Scholar) and in and The of the to of the relative expression of proteins a of and sites of phosphorylation from the brain regions and from the experimental we the of by proteins for which we identified more with a of than an of with a of greater than an of than with an of at as a and overall these an of relative to their protein A of were identified and protein with of the proteins identified by more the of protein an we the for protein for proteins with more The for this proteins with a have a protein of than that overall protein level a level of A of and to the by the PSD as as from in the of used in the independent in a of of which were identified in the a of 2159 identified in using a of A of 1564 sites of phosphorylation with the previously were in at of these were in both biological in both with at A of phosphorylation sites were in both relative to the of proteins is to the that is to a protein proteins to be identified using the in is of the that sites of phosphorylation to be on abundance proteins of and protein replicates, we the at the level of a in this and were identified in the replicates, and the relative of protein abundance the for both biological proteins identified in both the of protein the for of identified in both experiments a of proteins that from to the A of protein in in the a degree of and the for the of proteins the revealed that of were (e.g. be to from to a to the of PSD we proteins in the as at the M. D. H. K. A. S. J.C. M. for the of The 2000; PubMed Scopus Google Scholar). this of proteins that on a level of synaptic proteins with a of for these protein and an of a of we used proteins and on that region-specific expression The of expression levels consistent with findings and the of from experiments have used to that expression can be for of specific functional D. J. S. E.S. T.R. of expression with self-organizing methods and to Acad. Sci. S. A. PubMed Scopus Google Scholar). from brain in their expression of protein functional we used analysis to functional by their expression patterns. were organized at a level to different expression levels were consistent between both brain with the a cluster of proteins that a expression in the and higher expression in the We proteins of different were these by protein to its molecular function the of proteins to specific protein functional analysis revealed a strong of kinases and to be present in the cluster highest expression in the hippocampus the proteins as to cluster of of the proteins coordinated of region-specific protein expression, we used to proteins on the basis of of expression in brain the of methods using 2002; PubMed Scopus Google Scholar, J. J. cluster in 21: PubMed Scopus Google Scholar). The were both by brain regions and at the level of preparations a of J. 2006; PubMed Scopus Google Scholar). these for a of proteins with highest expression in the with the level of in the and in brain were to be the to other cluster glutamate receptor receptor and A cluster is by proteins with highest expression in the with a distinct expression of these proteins are in a in local protein synthesis and synaptic Neurosci. 2006; 26: PubMed Scopus Google Scholar), this from of function expression in the PSD preparations known are to be present in the is to that for the of in of A.J. M.D. M. M.P. a for between specific and protein PubMed Scopus Google Scholar, B. M. M. A proteomics to functional to 2003; 21: PubMed Scopus Google Scholar). an of a cluster of proteins with and expression, the NMDA receptor previously to be highly in the hippocampus and H. R. Schoepfer R. A. M. H. N. B. NMDA molecular and functional of PubMed Scopus Google Scholar, T. N. H. K. K. H. H. T. M. M. Molecular of the NMDA receptor 358: PubMed Scopus Google Scholar, NMDA receptor expression in the rat a analysis of levels of and Brain PubMed Scopus Google Scholar). of the in were of proteins in the as of this include with in the proteins in and NMDA receptor with in The that specific of proteins were synaptic expression in analysis that as a more be by for brain region-specific synaptic expression between proteins. this we used in the to the from the The between proteins in of these proteins of we determined the both and their These were groups the protein were in the as The overall of for higher than the of other protein were more to have greater than for Therefore, we that protein in that display highly expression previously as candidates for of protein with expression in brain regions that display highly expression were in the as We identified 1564 phosphorylation sites on proteins. In of the we were to the acid from the is to the acid that is a a of a of is relative to the to a as to a specific a phosphopeptide contains more than specific to be present in the to the assignment is for biological of specific more sites of phosphorylation were on proteins. we identified phosphorylation sites between the AMPA receptor from experiments protein expression and phosphorylation of In both replicates, relative phosphorylation highest in the hippocampus by cortex, and the overall for protein expression. relative phosphorylation levels by the relative protein expression relative phosphorylation that the level of phosphorylation highest in the hippocampus be by the that protein expression highest in the hippocampus. for and phosphorylation of the observed by In biological (e.g. when changes in the of a phosphorylation for by a can be to changes in protein level, changes in phosphorylation to the relative from of these us to synaptic preparations on a level by other In we the relative phosphorylation for from The for the hippocampus relative to the to which a relative in the hippocampus relative to the The both a higher relative phosphorylation when as a in the hippocampus relative to the the overall of phosphorylation and relative phosphorylation we determined the relative for phosphorylation sites in compare brain their relative can be as a the sites the in relative phosphorylation between the hippocampus and sites highest relative in the hippocampus were sites on and sites on In sites relative phosphorylation were greater in the than hippocampus a of sites of phosphorylation sites that display changes in between the and acid in a the overall regulation of phosphorylation brain we phosphorylation levels with to protein expression for proteins to be the of and of the brain from both biological as a the hippocampus a in the overall relative expression of with their and the relative expression brain regions of proteins as phosphatase kinase kinases identified with the is a of protein the and protein kinase highest expression in the hippocampus. In E.D. S. a in and a signaling Full Text Full Text PDF PubMed Scopus Google Scholar) and protein J.C. a protein kinase with to that regulates Neurosci. 2000; PubMed Google Scholar) highest expression in the cortex, and kinase S. of in the a of in PubMed Scopus Google Scholar) highest expression in the expression of a protein phosphatase protein kinase with its level of the expression of kinases and were and higher in the hippocampus relative to other brain regions and for kinases and phosphatases, that regulation of signaling a more role in the hippocampus than in other brain in the specific protein expression on proteins present in a of brain regions and on a of phosphorylation sites D. J. Duong D.M. J. T. Sheng M. J. and absolute of postsynaptic density from rat forebrain and Cell. Proteomics. 2006; 5: Full Text Full Text PDF PubMed Scopus (382) Google Scholar, R. M. M. D. D. B. R. and of protein phosphorylation in and Cell. Proteomics. 6: Full Text Full Text PDF PubMed Scopus Google Scholar). In this the to for and of both protein expression and phosphorylation levels from complex such as postsynaptic can from when is with can in more than In using the is by as the the long as the their the the of the for a in the of when with a we relative of 2159 proteins and 1564 sites of phosphorylation from the cortex, cerebellum, and hippocampus. The of proteins were identified and with to expression that overall were consistent with previously known expression patterns. proteins known to be highly in the cerebellum, the signaling and K. H. Y. M. expression of the glutamate receptor in PubMed Scopus Google Scholar, A.B. glutamate receptors are regulated PubMed Scopus Google Scholar, Y. A. M. R. N. S. and expression of rat glutamate receptors, and Neurosci. PubMed Google Scholar, H. R. A. The rat and the acid receptor PubMed Scopus Google Scholar, N. M. Y. K. expression and of protein of in the Biol. PubMed Scopus Google Scholar, of signaling region-specific expression of in rat Neurosci. 1997; PubMed Google Scholar, Y. A. S. K. T. expression of proteins in the 2004; PubMed Scopus Google Scholar, A. T. N. expression of proteins in rat J. PubMed Scopus Google Scholar), were observed the proteins with highest relative expression in the findings in the the extensive of synaptic protein and phosphorylation allowed us to of synaptic from a with more we that proteins known to a level of expression and suggest that protein correlating highly in this are known to In the to the synaptic expression of kinases and and 1564 sites of phosphorylation allowed us to for of signaling by the hippocampus relative to other brain The brain regions in this are from in their are known to be composed of different of neurons. a the of distinct synaptic is to be between distinct of synaptic to for by a of PSD components. We observed that known protein on a degree of expression brain of these have highly expression. exist for this proteins that highly synaptic expression the of of protein are in these specific a specific protein is associated with distinct synaptic its overall expression may with of the proteins in these is that protein with at In we of proteins expression were highly candidates for These proteins with expression in the brain regions that to have previously to a synaptic protein be to using a of (e.g. and and to the in which protein expression and post-translational levels are regulated biological The of this are a that and have allowed us to specific protein groups in analysis of protein groups both from the and allowed for the of that are such we AMPA receptors and the transmembrane AMPA receptor proteins are scaffolding that a role in the synaptic localization of AMPA AMPA receptors are composed of We observed a between expression of and consistent with the that the are a synaptic the in expression of highly with and A between and to a between and specific of the in with and expression and function of these S. in a with a Acad. Sci. S. A. PubMed Scopus Google Scholar, K. M. K. M. M. of AMPA receptor function in of Neurosci. 19: PubMed Google Scholar, N. K. H. S. S. controls AMPA receptor and synaptic Neurosci. PubMed Scopus Google the between the glutamate receptors AMPA and other AMPA as as proteins to with AMPA in a to the NMDA and AMPA receptor phosphorylation we of for of the NMDA receptor relative to the AMPA receptor both replicates, suggest as NMDA and AMPA receptor are present in in with a in the D. J. Duong D.M. J. T. Sheng M. J. and absolute of postsynaptic density from rat forebrain and Cell. Proteomics. 2006; 5: Full Text Full Text PDF PubMed Scopus (382) Google Scholar). we observed more for NMDA receptors H. C.G. A. analysis of from PubMed Scopus Google Scholar) with AMPA receptors M.B. Signal-processing machines at the postsynaptic density.Science. 2000; 290: 750-754Crossref PubMed Scopus (657) Google Scholar), consistent with the central role of NMDA receptors in and processing synaptic transmission patterns. regulation of protein phosphorylation an role in synaptic plasticity J. H. H. The molecular basis of function in synaptic and Rev. Neurosci. 2002; PubMed Scopus Google Scholar). an overall on region-specific in phosphorylation, we the of and from proteins to be in analysis that the level of phosphorylation highest in the hippocampus. this we protein expression of kinases and brain regions and that the levels of both of these protein are highest in the hippocampus these findings that the signaling is more in the hippocampus, that these a more role in synaptic plasticity than in other brain the activation of kinase signaling changes in phosphorylation at specific sites are on the of to a The from between the specific kinases and Therefore, a role for signaling in synaptic plasticity a biological for the central role that the hippocampus in term memory and its we present the synaptic protein expression and phosphorylation on a an is in identifying coordinated changes in synaptic composition and changes in synaptic function by and that are to a protein of phosphorylation in We for on the with