Organelle Proteomics of Rat Synaptic Proteins: Correlation-Profiling by Isotope-Coded Affinity Tagging in Conjunction with Liquid Chromatography-Tandem Mass Spectrometry to Reveal Post-synaptic Density Specific Proteins Ka wan Li,*,† Martin P. Hornshaw,‡ Jan van Minnen,† Karl-Heinz Smalla,§ Eckart D. Gundelfinger,§ and August B. Smit† Department of Molecular and Cellular Neurobiology, Center of Neurogenomics and Cognitive Research, Faculty of Earth and Life Sciences, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands, Applied Biosystems, Lingley House, 120 Birchwood Boulevard, Warrington, Cheshire, WA3 7QH, Great Britain, and Leibniz Institute for Neurobiology, Department of Neurochemistry and Molecular Biology, Brenneckestr. 6, D-39118 Magdeburg, Germany Received November 5, 2004
Organelle proteomics is the method of choice for global analysis of cellular proteins. However, it is difficult to isolate organelles to homogeneity. Recently, correlation-profiling has been used to filter off the contaminants ad hoc and to disclose the genuine organelle-specific proteins. In the present study, we further extend the method to include subcellular compartments that contain proteins shared by multiple distinct subcellular domains. We performed correlation profiling of proteins contained in synaptic membrane and postsynaptic density (PSD) fractions isolated from rat brain. Proteins were labeled with isotope-coded affinity-tag reagents, digested with trypsin, and resulting peptides were resolved by cation exchange chromatography followed by reversed phase chromatography. Peptides were then subjected to mass spectrometry for quantification and identification. We confirm that the core PSD proteins were enriched in the PSD preparation. Other functional protein groups such as cytoskeleton-associated proteins, protein kinases and phosphatases, signaling components and regulators, as well as proteins involved in energy production partitioned to multiple organelles. When analyzed as groups, they were shown to accumulate to a lesser extent. Mitochondrial proteins and transporters were generally strongly depleted from the PSD fraction confirming that they were contaminants of the PSD preparation. Finally, immunoelectron microscopy was performed on selected proteins to validate the proteomics results, and confirm that synaptophysin that was highly depleted in the PSD preparation is localized in the presynaptic compartment, whereas LASP-1 that was slightly enriched in the PSD preparation is present in the PSD as well as other subdomains within the synapse. Keywords: synapse • postsynaptic density • ICAT • MALDI TOF/TOF MS • rat
Introduction Organelle proteomics is an emerging approach for the global analysis of cellular proteins.1-3 It reduces the complexity of the samples allowing for the determination of the abundance of proteins in a given organelle preparation. Furthermore, because organelle proteomics focuses on the subcellular localization of proteins, it permits the modeling of organelle-specific protein interaction networks, and might give important clues to the physiology of the organelle.4,5 This knowledge can be used to formulate hypotheses for further in depth functional studies and to understand pathological states. * To whom correspondence should be addressed. Tel: 31-20-5987107. Fax: 31-20-5987112. E-mail:
[email protected]. † Vrije Universiteit. ‡ Applied Biosystems. § Leibniz Institute for Neurobiology. 10.1021/pr049802+ CCC: $30.25
2005 American Chemical Society
A prerequisite for organelle proteomics is to obtain the organelle in high purity. Toward this end multistep isolation techniques have been developed, which enrich the organelle of interest to a high degree.6 Nevertheless, it remains difficult, if not impossible, to totally exclude contaminants from preparations. Alternatively, correlation profiling has been proposed as the method of choice to filter out ad hoc the contaminants present in the preparation.7 This method assumes that proteins, which are contained in the same organelle, would follow the same degree of enrichment during the successive purification steps. This approach should greatly simplify the global analysis of protein constituents in the structurally well-defined organelles such as mitochondria, the Golgi apparatus, and the nucleus. On the other hand, many distinct subcellular compartments share overlapping sets of proteins, in which cases correlation profiling potentially may yield ambiguous results. Journal of Proteome Research 2005, 4, 725-733
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research articles In the present study, we demonstrate that even in such cases correlation-profiling can still yield meaningful insights into the organelle organization. Specifically, we used the isotope-coded affinity tag (ICAT) based technique for correlation profiling7,8 of the postsynaptic density (PSD), a subsynaptic organelle, from synapses of rat brain. The PSD is a compact electron-dense structure located at the postsynaptic compartment of the glutamatergic excitatory synapses.9 The protein constituents of the PSD play major roles in synaptic communication in the brain. Recent studies have indicated that the PSD is a dynamic organelle. Its morphology and protein content can be altered in a neuronal activitydependent manner,10,11 which is considered to be the molecular basis that regulates synaptic efficacy and plasticity, which in turn may underlie higher order brain functions such as learning and memory. To gain better insight into the structural and functional organization of the PSD several labs including ours’ have examined the proteome of the PSD thereby identifiying hundreds of proteins.12-15 In addition to previously identified bona fide PSD proteins, a number of additional proteins have been identified that can be grouped into various functional classes. These include proteins involved in membrane trafficking, protein synthesis, energy production, and protein folding and others. In view of the presence of these diverse groups of proteins in the PSD fraction, we proposed that the PSD may have the ability to function (semi-)autonomously and that it might direct various cellular functions in order to integrate synaptic physiology.14 However, a number of apparent contaminants such as mitochondrial proteins12-15 and even nuclear proteins13 were also detected. This raises the possibility that some functional groups previously detected in the PSD may constitute contaminants. In the present study, we have used ICAT-based correlationprofiling to gain insight into the degree of enrichment of PSD proteins as compared to the proteins of the synaptic membrane protein fraction. We confirm that the core PSD proteins were enriched in the PSD preparation. Other groups of proteins with various functions such as cytoskeleton-associated proteins, protein kinases and phosphatases, components and regulators of signaling pathways, and proteins involved in energy production may be associated with multiple organelles and multiprotein complexes, and consequently as groups they were enriched in the PSD fraction to a lesser extent. Mitochondrial proteins and transporters were generally strongly depleted indicating that they were likely contaminants of the PSD preparation. Finally, immunoelectron microscopy of a protein that was highly depleted in PSD preparation and a protein that was slightly enriched confirmed the differential localization of the proteins in the PSD.
Materials and Methods Isolation of Synaptic Membrane and PSD Protein Fractions. Synaptic membranes and PSDs were isolated as described previously.14 In brief, forebrains of rats were homogenized in 320 mM sucrose and then centrifuged at 1000 × g for 10 min. The supernatant was spun for 20 min at 12 000 × g. The pellet was suspended in 320 mM sucrose, loaded on top of a sucrose step gradient and centrifuged to obtain synaptosomes. Synaptosomes were lysed in hypotonic solution and the resulting synaptic membranes were recovered by centrifugation using another sucrose step gradient. To obtain the PSD protein fraction, synaptic membranes were extracted twice with Triton-X 100 to solubilize membrane proteins. The PSD is 726
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insoluble under these conditions and was recovered as intact organelle by sucrose gradient centrifugation. ICAT Labeling, Proteolysis and Manual Fractionation of the Samples. Labeling and tryptic digestion of protein fractions were carried out as described previously.14 Briefly, the samples were dissolved in 1.5% SDS in 50 mM Tris-HCl, pH 8. Protein concentrations were determined using Bradford measurement. The PSD and synaptic membrane proteins were labeled with cleavable ICAT heavy and light reagents, respectively, according to the standard Applied Biosystems ICAT protocol. After pooling the samples they were diluted to 0.1% SDS, and then incubated with modified trypsin for 16 h at 37 °C. The digested sample was diluted with “Cation Exchange Buffer-Load” to pH 3, and injected into the Cation-Exchange Cartridge. After washing, the bound peptides were eluted in four consecutive fractions using 100 mM KCl, 200 mM KCl, 300 mM KCl and finally 400 mM KCl in 10 mM KH2PO4, pH 3, 25% acetonitrile. The cationexchange fractions were neutralized and injected onto the avidin cartridge separately. The bound peptides were eluted with “Affinity Buffer-Elute”, dried in a speedvac, incubated for 2 h at 37 °C with cleaving reagents, and then dried again in a speedvac. The samples were dissolved in 60 µL 0.1% TFA, and the peptides separated by capillary reversed-phase HPLC. Capillary HPLC and Mass Spectrometry. Peptides were delivered with a FAMOS autosampler (LC-packing) at 30 µL/ min to a C18 trap column (1 mm × 300 µm i.d. column) and separated on an analytical capillary C18 column (150 mm × 100 µm i.d. column) at 500 nl/min, using the LC-packing Ultimate system. The peptides were separated using a linearly increasing concentration of acetonitrile from 5 to 50% in 30 min, and to 100% in 5 min. The eluent was mixed with matrix (7 mg R cyano-hydroxycinnaminic acid in 1 mL 50% acetronitrile, 0.1% TFA, 10 mM dicitrate ammonium) delivered at a flow rate of 1.5 µL/min, and deposited off-line to the Applied Biosystems stainless steel sample plate every 15 s for a total of 192 spots using a fraction collecting robot, the Probot from Dionex. Both MS and MS/MS measurements of the peptides were carried out on an Applied Biosystems 4700 Proteomics Analyzer with TOF/TOF optics as described previously.14 The daughter ion spectra were searched against the Celera Discovery System (CDS) database, using the Mascot (Matrix Science) search engine embedded within the GPS Explorer v2.0 (Applied Biosystems) software program installed locally. GPS Explorer was also used to perform the ICAT quantitation experiments. The taxonomy for the database search was rodent. Immuno-Electron Microscopy. For immuno-electron microscopy, rats were perfused with 2% paraformaldehyde and 0.2% glutaraldhyde in PBS. The brain was dissected and postfixed in the same fixative for 2 h, and next transferred to PBS. After 24 h, the hippocampus (CA1-CA3 region) was dissected, and cut into 1 mm3 cubes. These were immersed with 2.4 M sucrose at 4 °C overnight, mounted on aluminum rivets and quickly frozen, and next stored in liquid nitrogen until use. 70 nm ultrathin cryosections were cut at -120 °C on a Reichert cryo-ultramicrotome and incubated for 1 h with the primary antiserum (rabbit-anti-synaptophysin; Sanver Tech.) and goatanti-LASP1 (Abcam), both diluted 1:500 in PBS. For antisynaptophysin, sections were subsequently incubated with 1 nm gold-conjugated goat-anti-rabbit Fab fragments (dilution 1:100 in PBS; Aurion). After washing, the gold particles were intensified with R-Gent SE EM silver enhancement kit (Aurion) according to the manufacturer’s instructions. For anti-Lasp1, sections were incubated (45 min) with rabbit-anti-goat, washed
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Correlation-Profiling of Organelle Proteomics
in PBS. Next, sections were incubated in gold conjugated (10 nm) Protein A (45 min) and washed in PBS. The sections were subsequently mounted in a mixture of 0.4% methylcellulose and 1.8% uranyl acetate. After drying, the sections were viewed with a JEOL 1010 electron microscope.
Results Samples containing ICAT-labeled proteins of the synaptic membrane fraction (light ICAT modification) and the PSD fraction (heavy ICAT modification) were digested with trypsin and fractionated into 4 cation exchange fractions. Each fraction was further separated by nano-reversed phase chromatography and the eluent collected off-line onto 192 spots of a single stainless steel MALDI sample plate and analyzed by MALDI MS and MS/MS. Peptides with a minimum signal-to-noise ratio of 60 (noise actually equals the root-mean-square of the noise) from the single stage MS analysis were selected automatically for MS/MS analysis. A maximum of 15 MS/MS spectra were allowed per spot. In total, approximately 5000 MS/MS acquisitions were performed. Peptides that were not labeled by ICAT were excluded from the analysis. A number of synaptic proteins were completely depleted from the PSD preparation, and appeared as singletons in the MS analysis. These proteins are considered as contaminants and not discussed further. To increase identification stringency we selected only those proteins identified with a Mascot best ion score for an individual peptide above 20, and a total ion score confidence interval percentage of at least 95% (calculated by the GPS Explorer software) for identification. The great majority of this class of identifications scored in excess of 99%. In total, 129 ICAT pairs fulfilled these criteria (Table 1). 60 ICAT pairs have an ICAT ratio above 1 implying that they were enriched in the PSD preparation. As expected, a large number of identified proteins have an ICAT ratio substantially below 1, which may constitute the “contaminants” or proteins loosely associated with the PSD. Interestingly, there were also proteins that seem to distribute equally in synaptic membrane and PSD preparation or were only slightly reduced in the PSD preparation, suggesting that they might be present within as well as outside the PSD. To gain a better insight into the differential localization of protein classes, individual proteins are grouped based on their function, and in the specific case of proteins contained in mitochondria they are grouped based on their mitochondrial origin. Figure 1 shows the ICAT ratio of functional groups of proteins from the PSD preparation versus synaptic membrane. To obtain a meaningful trend of protein distribution among the subcellular compartments it is necessary to have a reasonable number of members per group; therefore, we focus on functional groups that contained at least three distinct members. The clustering of functional groups reveals the existence of several distribution patterns. In agreement with previous studies, scaffolding proteins, cytoskeletal proteins, and ion channels/ receptors were particularly enriched in the PSD. The functional group of signaling components and regulators seems to fall into three sub-groups; one that was 2-3 times enriched in the PSD, a second one that might be equally distributed in the PSD and synaptic membrane, and a third group that was depleted from the PSD fraction. Protein kinases and phosphatases, and cytoskeleton-associated proteins displayed a gradual transition of individual group members from those enriched within the PSD preparation to those depleted from it. The G-protein coupled receptors, which were detected in
this study, distributed equally between the PSD and synaptic membrane. On the other hand, most of the identified proteins involved in energy production or membrane trafficking were under-represented in the PSD preparation, with only a few of them slightly enriched. Transporters and proteins involved in protein metabolism and modification were all depleted from the PSD preparation. Mitochondrial proteins constituted the biggest group of proteins present in the PSD preparation, and as expected basically all of them were depleted indicating that they are contaminants of the PSD fraction. Two novel (hypothetical) proteins were also enriched (see Table 1); one of them displayed a very high degree of enrichment suggesting that it is a genuine PSD protein. Their functions remain to be determined. To independently verify the distribution of the synaptic proteins, we have performed immunoelectron microscopy on two selected proteins, one that was depleted and the other slightly enriched in the PSD preparation. In agreement with our ICAT correlation profiling data, Figure 2 A shows that synaptophysin, a well established presynaptic protein, is located in the presynaptic compartment and associated mainly with the synaptic vesicles. On the other hand LASP-1 is localized both in the cytoplasmic side of the PSD and other synaptic structures (Figure 2B), suggesting that this protein may be partitioned among different sub-domains of the synapse.
Discussion Recently, we and several other groups have carried out studies to elucidate the proteome of the PSD. These studies identified hundreds of proteins that were contained in the PSD preparation.12-14 To confirm the enrichment of the novel PSD proteins in the PSD preparation, immunoblotting14,15 or mass spectrometry-based absolute quantitation with internal peptide standard12 were employed. While these approaches are useful to study a small number of proteins, they are not suitable for a global quantative analysis. In the present study, we perform the first correlationprofiling analysis of the PSD proteome. Previous studies have demonstrated the applicability of such an approach to discriminate genuine organelle proteins from contaminants.7 However, as the PSD has to be considered as an ‘open’ organelle, which can recruit and release proteins on demand, the distinction between bona fide PSD proteins and putative contaminants, which are carried from the synaptic membrane preparation into the PSD fraction, may not be as clear-cut as with the other structurally and functionally well defined organelles, such as the centrosome.7 Proteins with decreasing relative amounts in the PSD as compared to the synaptic membrane fraction may fall into different groups: (a) A subset of the PSD proteome is dynamically exchanged with other subcellular compartments of dendritic spines, some of them constitutively and others in an activity-dependent manner (see for example.16 (b) A number of endogenous PSD proteins may also have general functions throughout the neuron and therefore are present in many other subcellular compartments. (c) Different types of excitatory PSD-containing synapses can be discerned based on their morphology and function (e.g., spine synapses, shaft synapses, mossy fiber terminals). It is therefore expected that the constituents of the PSD from these synapses overlap, but may contain also synapse type-specific proteins. (d) True or potential contaminants may behave biochemically like PSD members and therefore may co-purify with the PSD Journal of Proteome Research • Vol. 4, No. 3, 2005 727
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Table 1. Identified ICAT Pairs from PSD and Synaptic Membrane Preparationsa
protein name
Maguin- 1 similar to Actin, cytoplasmic 1 hypothetical protein XP_242579 guanylate kinase associated protein neurofilament protein NF-H beta-spectrin 3 densin-1 Calcium/calmodulin-dependent protein kinase type II beta chain Similar to alpha internexin neuronal intermediate filament protein similar to hypothetical protein KIAA0522 (ArfGEF) channel associated protein of synapse 2 Creatine kinase Psd-95 similar to KIAA0763 gene product (SynArfGEF) Ca2+/calmodulin-dependent protein kinase II alpha MKIAA0777 protein (Arg/Abl-interacting protein) Microtubule-associated protein 2
accession no.
protein peptide pI count
molecular function
117317 10482 25163 74187 115213 270769 167584 60364
6.3 9.0 6.8 5.4 5.7 5.6 6.1 6.7
1 3 2 2 5 3 4 4
55349
5.4
3
rf|XP•228841.1
173878
7.6
3
pir|T10811 spt|P09605 pdb|1JXM•A rf|XP•232193.1
94832 47355 34735 139847
5.8 8.8 6.1 8.9
5 2 6 5
gb|AAH31745.1
54081
6.6
3
trm|Q80TS1
127651
8.4
2
trm|Q63724
198863
4.8
2
Glutamate [NMDA] receptor subunit zeta 1 precursor (NR1) Plectin 1 Glycogen synthase kinase-3 alpha
spt|P35439
105442
9.0
2
spt|P30427 spt|P18265
533214 50995
5.7 9.0
6 4
CPG2 protein
trm|Q63128
108969
5.7
4
SAP 102 MKIAA0059 protein (actin-binding LIM protein) protein kinase C gamma
trm|Q80TH1 trm|Q80U86
103767 75616
6.3 8.7
2 2
78307
7.3
6
SynGAP-b
trm|Q9QX02
137272
9.1
6
similar to hypothetical protein DKFZp434N1131.1 (E3 ubiquitin ligase) Calmodulin-binding protein
rf|XP•237138.1
20565
9.2
2
trm|Q63092
54072
5.4
3
Neuron-specific class III beta-tubulin similar to LIM-only protein 6 (prickle like 2)
trm|Q8K5B6 rf|XP•144905.2
50255 102168
4.9 8.1
4 2
Liprin alpha 3 Glial fibrillary acidic protein Myosin Va
trm|Q91Z79 trm|Q925K3 spt|Q9QYF3
115971 46498 211630
5.4 5.1 8.9
2 1 3
tubulin beta chain 15 - rat LIM and SH3 domain protein 1 (LASP-1)
pir|A25113 spt|Q61792
49905 29975
4.8 6.6
4 4
Serine/threonine protein phosphatase type 1 alpha Creatine kinase, ubiquitous mitochondrial precursor synaptic SAPAP-interacting protein Synamon GABA B receptor 1 g D-MeAsp receptor:SUBUNIT)epsilon2
trm|Q9Z1G2
37516
5.9
6
spt|P25809
46999
8.7
5
kinase/ phosphatase cytoskeleton cell adhesion/ polarity scaffolding glia cytoskeletonassociated cytoskeleton cytoskeletonassociated kinase/ phosphatase mitochondria
gb|AAD04569.2
225383
8.6
3
scaffolding
trm|Q920D8 prf|1814459A
25385 165477
9.2 6.4
4 8
rf|XP_232642.1 rf|XP•130243.2 trm|Q9Z2Y3 rf|XP•219468.1
152900 70231 41389 84893
6.0 8.8 5.3 8.5
3 2 1 2
GPC reeptor ion channel receptor ubiquitination novel scaffolding GPC reeptor
rf|XP•227473.1
12692
9.1
2
rf|XP•227804.1
45956
6.1
2
similar to KIAA0849 protein (cylindromatosis) RIKEN cDNA A930041I02 gene Vesl-1L similar to tumor endothelial marker 5 precursor similar to NADH dehydrogenase (ubiquinone) Fe-S protein 5 similar to adenylate kinase 5
728
trm|Q9Z1T4 rf|XP•148712.1 rf|XP•242579.1 gb|AAC53054.1 prf|1601423A rf|NP•062040.1 trm|Q80TE7 spt|P08413
protein MW
trm|Q8VCW5
emb|CAA30267.1
Journal of Proteome Research • Vol. 4, No. 3, 2005
total ion score
total ion best avg score ion ICAT C. I. % score ratio
scaffolding cytoskeleton novel scaffolding cytoskeleton cytoskeleton scaffolding kinase/ phosphatase cytoskeleton
95 55 50 58 116 140 96 165
100 100 100 100 100 100 100 100
95 22 37 40 45 125 49 67
5.56 4.08 3.88 3.77 3.62 3.42 3.28 3.21
45
100
26
3.17
signaling/ regulator scaffolding mitochondria scaffolding signaling/ regulator kinase/ phosphatase scaffolding
82
100
76
3.15
349 58 467 54
100 100 100 100
140 58 165 38
2.78 2.60 2.59 2.59
174
100
146
2.54
35
97
35
2.32
36
98
36
2.28
96
100
71
2.26
197 114
100 100
67 81
2.19 2.14
42
99
32
2.11
130 41
100 99
82 29
2.09 2.07
102
100
36
1.98
121
100
62
1.96
50
100
32
1.93
61
100
28
1.92
122 88
100 100
57 76
1.83 1.81
96 50 45
100 100 100
62 50 39
1.80 1.75 1.72
126 66
100 100
57 54
1.66 1.58
169
100
57
1.52
249
100
59
1.48
53
100
35
1.47
53 262
100 100
34 63
1.47 1.46
84 46 114 57
100 100 100 100
67 43 114 52
1.45 1.45 1.40 1.40
mitochondria
49
100
25
1.39
energy production
61
100
58
1.36
cytoskeletonassociated ion channel receptor cytoskeleton kinase/ phosphatase cytoskeletonassociated scaffolding cytoskeletonassociated kinase/ phosphatase signaling/ regulator ubiquitination
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Correlation-Profiling of Organelle Proteomics Table 1. (Continued)
protein name
accession no.
protein protein peptide MW pI count
similar to beta-catenin
rf|XP•235316.1
68945
5.8
3
Cyclic nucleotide phosphodiesterase 1
trm|Q923F3
47094
9.1
3
alphaII spectrin cAMP-regulated guanine nucleotide exchange factor II Rat brain 4.1(L)
emb|CAA62350.1 284420 rf|NP•062662.1 113417
5.2 6.4
4 2
trm|Q9WTP1
170906
5.2
3
GABA B receptor 2 similar to Coronin 2B
rf|NP•113990.1 105696 rf|XP•217182.1 16654
9.0 8.7
1 1
Voltage-dependent calcium channel gamma-8 subunit Vascular endothelial growth factor receptor 3 precursor similar to triple functional domain similar to cyclin-dependent kinase-like 1 (CDC2-related kinase) Jagged 2 precursor (Jagged2) Tubulin alpha-3/alpha-7 chain (Alpha-tubulin 3/7) Rabphilin-3A (Exophilin 1) protein 4.1
spt|Q8VHW2
43426
9.3
3
spt|P35917
152919
5.9
5
rf|XP•226888.1 rf|XP•233822.1
224527 66253
6.0 8.9
spt|Q9QYE5 spt|P05214
134637 49928
spt|P47708 gb|AAA37122.1
molecular function
cell adhesion/ polarity signaling/ regulator cytoskeleton signaling/ regulator cytoskeletonassociated GPC reeptor cytoskeletonassociated ion channel
total ion best avg total ion score ion ICAT score C. I. % score ratio
91
100
87 1.34
79
100
44 1.33
82 36
100 97
43 1.30 28 1.30
75
100
53 1.29
55 60
100 100
55 1.29 60 1.25
47
100
34 1.23
73
100
31 1.23
3 3
growth factor receptor signaling/regulator kinase/phosphatase
73 55
100 100
50 1.21 22 1.16
5.5 5.0
6 2
signaling/regulator cytoskeleton
51 36
100 98
26 1.11 35 1.09
75442 78801
8.6 6.0
3 3
163 73
100 100
117 1.05 47 1.05
trm|Q9QUL6 spt|P47754
82600 32947
6.6 5.6
5 1
150 43
100 99
53 1.00 43 0.86
55
100
47 0.79
82
100
71 0.79
41 63
99 100
31 0.78 50 0.77
34 93
96 100
23 0.77 93 0.69
57
100
36 0.66
36 62
98 100
30 0.64 52 0.63
70 37
100 98
33 0.61 32 0.59
58 34 117
100 96 100
52 0.51 31 0.50 106 0.48
100 104 60 107 98 40
100 100 100 100 100 99
100 68 32 58 98 38
184
100
146 0.30
207
100
58 0.29
N-ethylmaleimide sensitive factor F-actin capping protein alpha-2 subunit (CapZ alpha-2) similar to KIAA0672 gene product (RhoGAP domain) Microtubule-associated protein tau
rf|XP•220588.1
94928
6.5
2
trm|Q91WK4
38838
9.5
2
erk-1 Fructose-bisphosphate aldolase A
pir|S28184 spt|P05065
42766 39196
6.2 8.4
3 2
Myotubularin-related protein phosphoglycerate mutase
trm|Q8K3W5 dbj|BAB26576.1
57617 28685
6.6 6.7
3 1
glutamine synthetase (glutamateammonia ligase) stromal membrane associated protein, ArfGAP 14-3-3 zeta
rf|NP•058769.1
42259
6.8
2
trm|Q91VZ6 dbj|BAA13421.1
47630 27737
8.7 4.7
2 2
trm|P97582 rf|XP•124864.1
89927 11260
8.1 8.9
3 2
spt|P51881 gb|AAA40932.1 spt|P00507
32910 42699 47284
9.7 5.3 9.1
2 2 2
spt|O55125 trm|Q80TL4 emb|CAB41017.1 pir|A60347 emb|CAA32202.1 rf|XP•231706.1
33342 45578 58027 21436 61377 57582
9.5 5.7 5.7 8.8 8.1 7.2
1 3 3 3 1 2
rf|XP•215224.1
42805
9.2
2
trafficking cytoskeletonassociated trafficking cytoskeletonassociated signaling/ regulator cytoskeletonassociated kinase/phosphatase energy production kinase/phosphatase energy production protein metabolism/ modification signaling/regulator signaling/ regulator cytoskeleton protein metablosim/ modification mitochondria mitochondria protein metablosim/ modification trafficking novel transporter signaling/ regulator glia kinase/ phosphatase mitochondria
rf|XP•218624.1
102700
6.2
9
trafficking
rf|XP•223434.1 trm|Q9D6M3
33946 34648
9.8 9.3
2 3
mitochondria mitochondria
42 76
99 100
35 0.29 33 0.29
gb|AAD22722.1 trm|Q8C2J5 spt|Q91WS0
29777 103951 12089
9.1 6.5 9.2
3 6 4
mitochondria trafficking novel
96 140 167
100 100 100
59 0.28 65 0.28 60 0.27
68283
5.4
3
transporter
45
100
25 0.27
Ankyrin similar to Peptidyl-prolyl cis-trans isomerase A ADP,ATP carrier protein, fibroblast isoform creatine kinase Aspartate aminotransferase NipSnap1 protein MKIAA1045 protein SERCA2a isoform GTP-binding protein rac1 glutamate dehydrogenase similar to RIKEN cDNA 2610037M15 (lipid kinase) similar to NAD+-isocitrate dehydrogenase, gamma subunit similar to adaptor-related protein complex AP-2, alpha 1 subunit similar to CG7943 1300006L01Rik protein (Weakly similar to solute carrier) AF048829_1 voltage dependent anion channel Adaptor protein complex AP-2 Uncharacterized hematopoietic stem/progenitor cells protein MDS029 homolog ATPase, H+ transporting, lysosomal 70kD, V1 subunit A, isoform 1
trm|Q8CHX2
0.45 0.40 0.35 0.34 0.32 0.31
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Table 1. (Continued) protein MW
protein pI
peptide count
molecular function
total ion score
total ion score C. I. %
best ion score
avg ICAT ratio
spt|Q9DCJ5
19848
8.8
1
mitochondria
85
100
85
0.25
rf|XP•146386.1
49435
7.7
2
mitochondria
164
100
89
0.25
trm|Q62634
61625
7.0
1
transporter
59
100
59
0.23
rf|XP•224830.1 trm|Q8BH59 prf|2001428A rf|XP•217417.1
31333 74523 67526 45544
4.9 8.4 6.5 5.1
1 4 4 2
trafficking mitochondria trafficking mitochondria
37 148 189 56
98 100 100 100
37 104 116 33
0.22 0.22 0.21 0.21
rf|XP_227798.1 trm|Q80WP4 spt|P35571
105644 86074 80921
9.0 6.4 6.2
4 2 2
37 42 83
98 99 100
21 34 66
0.21 0.20 0.19
dbj|BAA83105.1 132502 spt|P47942 62239
5.8 6.0
3 1
55 67
100 100
40 67
0.18 0.18
rf|XP•192845.1
18495
7.5
2
54
100
38
0.16
CYPH_RAT Peptidyl-prolyl cis-trans isomerase A
sp|P10111
17863
8.3
3
116
100
52
0.15
Clathrin heavy chain Ubiquinol-cytochrome C reductase complex 11 kDa protein 1110002H15Rik protein similar to heat shock protein 60 ATPase, Na+K+ transporting, alpha 3 subunit NADH-ubiquinone oxidoreductase 42 kDa subunit vacuolar adenosine triphosphatase subunit B pyruvate kinase
trm|Q80U89 spt|P99028
192208 10428
5.4 4.8
5 3
novel mitochondria energy production transporter cytoskeletonassociated cytoskeletonassociated protein metablosim/ modification trafficking mitochondria
342 208
100 100
121 117
0.14 0.14
trm|Q9CR61 rf|XP•232938.1 trm|Q8VCE0
16320 108095 115894
8.4 8.2 5.4
1 3 8
mitochondria chaperone transporter
50 84 247
100 100 100
50 43 73
0.13 0.13 0.13
spt|Q99LC3
40578
7.6
2
mitochondria
94
100
55
0.12
gb|AAC52411.1
56549
5.6
4
transporter
222
100
109
0.10
pir|B26186
57781
6.6
3
109
100
55
0.10
similar to Acyl carrier protein Guanine nucleotide-binding protein G(i)
rf|XP•215044.1 spt|P38401
17503 40224
4.9 5.7
2 4
51 82
100 100
50 55
0.10 0.09
vacuolar proton-translocating ATPase 100 kDa subunit isoform a1-I hypothetical protein XP_159306 mGLT-1B Synaptic vesicle protein 2B similar to Protein CGI-51 hexokinase Similar to NADH dehydrogenase cytochrome c 1 NAD+-specific isocitrate dehydrogenase a-subunit similar to dihydrolipoamide dehydrogenase similar to mitochondrial voltage dependent anion channel Synaptophysin pyruvate dehydrogenase (lipoamide) phosphopyruvate hydratase
gb|AAF59918.1
96231
6.1
2
energy production mitochondria signaling/ regulator transporter
56
100
30
0.09
rf|XP•159306.1 13803 dbj|BAA23772.1 60577 trm|Q63564 77451 rf|XP•217009.1 16186 pir|A35244 102207 trm|Q91YT0 50802 dbj|BAB22380.1 35418 trm|Q99NA5 39588
7.9 6.2 5.4 8.8 6.5 8.5 9.3 6.5
2 2 3 2 2 1 2 2
novel transporter pre-synaptic mitochondria mitochondria mitochondria mitochondria mitochondria
42 73 58 58 106 58 275 89
100 100 100 100 100 100 100 100
29 69 26 43 75 58 180 87
0.08 0.08 0.08 0.07 0.06 0.06 0.06 0.05
rf|XP•216682.1 rf|XP•212659.1
54004 30737
8.0 8.6
1 3
mitochondria mitochondria
71 308
100 100
71 169
0.05 0.04
trm|Q91WI8 pir|DERTP1 pir|JC1039
33346 43169 47096
4.9 8.4 5.1
2 5 2
93 93 145
100 100 100
91 36 127
0.04 0.04 0.04
Cytochrome c oxidase, subunit Vb
trm|Q9D881
13838
8.3
1
pre-synaptic mitochondria energy production mitochondria
38
98
38
0.03
protein name
NADH-ubiquinone oxidoreductase 19 kDa subunit similar to Elongation factor Tu, mitochondrial precursor Brain specific Na+-dependent inorganic phosphate cotransporter similar to glycoprotein M6A Solute carrier family 25 syntaxin-binding synaptic protein similar to NADH-ubiquinone oxidoreductase 75 kDa similar to hypothetical protein FLJ20354 Mitochondrial assembly regulatory factor Glycerol-3-phosphate dehydrogenase plasma membrane Ca2+-ATPase isoform 2 Dihydropyrimidinase related protein-2 (TOAD-64) cofilin 1, nonmuscle
accession no.
a
MS/MS spectra were searched against the CDS combined database, using the mascot search engine within the GPS Explorer software. All of the identified proteins listed have a total ion score confidence interval percentage of greater than 95%.
fraction. An example of this kind may be the glial intermediate filament protein, GFAP. Indeed, our analysis reveals the different degrees of enrichment among the distinct functional groups of proteins in the PSD preparation that seem to reflect these co-purification patterns. As expected glutamate receptors, which are the 730
Journal of Proteome Research • Vol. 4, No. 3, 2005
molecular signature of the postsynaptic compartment, were enriched in the PSD fraction.17 Many scaffolding proteins are known to be integral components of the PSD and accordingly constitute the most enriched group of PSD proteins. These proteins anchor the glutamate receptors in the PSD, and link distinct protein complexes to form functional units. They
Correlation-Profiling of Organelle Proteomics
research articles
Figure 1. ICAT ratio of proteins contained in PSD preparation vs synaptic membrane protein fraction. Proteins are grouped according to their function. The individual columns represent individual proteins ratios from the ICAT profiling as stated in Table 1.
recruit signaling components and regulators into the PSD. Finally, scaffolding proteins often connect to cytosketelal proteins to maintain or alter spine morphology.18 Cytoskeletal proteins are another functional group that shows enrichment with the PSD fraction. Although these structural proteins are present throughout the cytoplasm of cells, they are particularly concentrated in the dendritic spine and form a dense network that maintains and regulates the spine morphology and the trafficking of the protein complexes across the synaptic subdomains. However, a number of cytoskeletal elements may also be enriched in the PSD fraction because of their biochemical
characteristics (see above). Cytoskeleton-associated proteins, on the other hand, constitute a more heterogeneous group of proteins with respect to their PSD association; some of them were enriched whereas others were depleted. It is known that a subset of cytoskeleton-associated proteins play major roles as regulators of cytokeleton dynamics. Inspection of this class of proteins reveals that some of them were enriched (Lasp-1), some showed no change (F-actin capping protein) and others were under-represented (cofilin) in the PSD fraction. Members of this class of proteins may be present in most subcellular domains where cytoskeletal proteins are located, and could be Journal of Proteome Research • Vol. 4, No. 3, 2005 731
research articles
Figure 2. Immunoelectron microscopy of a synapse. A was stained with anti-synaptophysin, B with anti-Lasp1. In A gold particles are associated with the synaptic vesicles (SV), and not with the PSD (arrows). In B, Lasp1 immunoreactivity is in close contact with the PSD, whereas there is no labeling of the synaptic vesicles in the presynaptic terminal (Pr). Arrowhead indicates the presence of immunoreactivity outside the PSD. Po; postsynaptic terminal.
recruited to synaptic membrane subdomains including the PSD in an activity dependent manner.11 This is supported by our immunoelectron microscopy studies, which shows that LASP-1 is present in the PSD as well as other synaptic compartments. Accordingly, the ICAT ratios of cytoskeleton-associated proteins may reflect the abundance of these proteins in different subdomains of the synaptic membrane under a given condition. In agreement with previous studies, we show that mitochondrial proteins were greatly reduced or almost nonexistent in the PSD preparation as compared to the synaptic membrane fraction. One notable exception is the mitochondrial creatine kinase, which was enriched in the PSD preparation. The function of creatine kinase is the transport of energy from mitochondria to other subcellular compartments, especially those that have a high demand for energy. As the physiological processes in the PSD, such as ion transport, signaling, protein synthesis, protein trafficking and ubiquitination are energy demanding, the large amount of creatine kinase transported to the PSD might form an important source of energy supply. Eventually, it has to be tested whether this enzyme can shuttle from mitochondria to other subcellular compartments such as the PSD. Proteins involved in signal transduction cascades are known to regulate the physiology and morphology of the synaptic spine, and play important roles in the induction and maintenance of neuronal plasticity.19 It is generally agreed upon that the physiological functions of signaling proteins, e.g., protein kinases, which often distribute in multiple subcellular compartments, are critically dependent on the precise location of the proteins. Among the large number of protein kinases and phosphatases, some of them were reported to be clustered within the PSD and spatially positioned for the regulation of localized signaling cascades.19 The present study demonstrates that a number of these kinases and phosphatases, such as CaMKII, serine/threonine protein phosphatase, CDC2-related kinase, glycogen synthase kinase and protein kinase C, were enriched in the PSD fraction, whereas ERK-1 partitioned equally to the PSD and the synaptic membrane. This suggests that this latter protein may be involved in the functioning of PSD as well as other micro-domains located elsewhere near the synaptic membrane. G protein-coupled receptors, small GTPases and their regulators were also present in the PSD preparation; their partitioning between PSD and synaptic membranes seems to be receptor type-specific. In agreement 732
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Li et al.
with previous studies,20,21 we demonstrate that SynArfGEF and SynGAP were enriched in the PSD preparation, while on the other hand the stromal membrane-associated protein, ArfGAP, was under-represented. There are several classes of proteins represented by only a few members. Proteins involved in ubiquitination were generally enriched, which is in agreement with the role of this process in the regulation of synaptic efficacy.22,23 Members of proteins involved in membrane trafficking were either equally distributed in the PSD and synaptic membrane or are underrepresented in the PSD preparation. As protein trafficking is an integrated process for the functioning of all organelles, proteins involved in it must be present across all subcellular compartments, which corresponds to the fact that they are not enriched in the PSD. Proteins involved in protein metabolism and protein transporters were highly under-represented and may likely represent loosely associated PSD components or even contaminants. Finally, we identified several novel proteins enriched in PSD, the identity of which have to be investigated in more detail. It should be noted that ICAT reagents label cysteine-containing proteins. Therefore, synaptic proteins that do not contain cysteine will not be detected. Alternative reagents such as the recently developed iTRAQ, that labels N-termini and the lysine side chain, may prove to be even more useful for the detection and quantification of synaptic proteins.24 Taken together, the present correlation-profiling study yields a global view on the organization of the PSD. The core proteins of the PSD such as scaffolding proteins and some cytoskeletal proteins were highly enriched in PSD preparations and mitochondrial proteins and transporters were strongly diminished. This is in accordance with the assumption that genuine organelle proteins should be enriched and contaminants should be depleted. Interestingly, there are groups of proteins that are less clear-cut in their spatial distribution, and may partition into multiple subdomains. This is in line with the current model that considers the PSD as an ‘open’ organelle integrated into the synaptic spine, which may function in a semi-autonomous manner.14 Alternatively, it can be argued that this group of proteins does not follow the normal correlationprofiling pattern and therefore their spatial localization is dubious. Temporal staged correlation-profiling of these proteins during events that induce alteration of protein trafficking between the PSD and other subdomains of synaptic membrane will be useful to better define the dynamics of their spatial distribution.
Acknowledgment. The authors thank Center of Medical Systems Biology, Netherlands, for its financial support. K.H.S. and E.D.G. are supported by a grant from the Land SaxonyAnhalt and the Fonds der Chemischen Industrie. References (1) Kikuchi, M.; Hatano, N.; Yokota, S.; Shimozawa, N.; Imanaka, T.; Taniguchi, H. J. Biol. Chem. 2004, 279, 421-428. (2) Blondeau, F.; Ritter, B.; Allaire, P. D.; Wasiak, S.; Girard, M.; Hussain, N. K.; Angers, A.; Legendre-Guillemin, V.; Roy, L.; Boismenu, D.; Kearney, R. E.; Bell, A. W.; Bergeron, J. J.; McPherson, P. S. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 3833-3838. (3) Warnock, D. E.; Fahy, E.; Taylor, S. W. Mass Spectrom. Rev. 2004, 23, 259-280. (4) Brunet, S.; Thibault, P.; Gagnon, E.; Kearney, P.; Bergeron, J. J.; Desjardins, M. Trends Cell Biol. 2003, 13, 629-638. (5) Grant, S. G. Bioessays 2003, 25, 1229-1235. (6) Huber, L. A.; Pfaller, K.; Vietor, I. Circ. Res. 2003, 92, 962-968. (7) Andersen, J. S.; Wilkinson, C. J.; Mayor, T.; Mortensen, P.; Nigg, E. A.; Mann, M. Nature 2003, 426, 570-574.
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(17) Kennedy, M. B. Science 2000, 290, 750-754. (18) Garner, C. C.; Nash, J.; Huganir, R. L. Trends Cell Biol. 2000, 10, 274-280. (19) Sheng, M.; Kim, M. J. Science 2002, 298, 776-780. (20) Inaba, Y.; Tian, Q. B.; Okano, A.; Zhang, J. P.; Sakagami, H.; Miyazawa, S.; Li, W.; Komiyama, A.; Inokuchi, K.; Kondo, H.; Suzuki, T. J Neurochem. 2004, 89, 1347-1357. (21) Kim, J. H.; Liao, D.; Lau, L. F.; Huganir, R. L. Neuron 1998, 20, 683-691. (22) Ehlers, M. D. Nat. Neurosci. 2003, 6, 231-242. (23) Colledge, M.; Snyder, E. M.; Crozier, R. A.; Soderling, J. A.; Jin, Y.; Langeberg, L. K.; Lu, H.; Bear, M. F.; Scott, J. D. Neuron 2003, 40, 595-607. (24) Ross, P. L.; Huang, Y. N.; Marchese, J. N.; Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.; Pillai, S.; Dey, S.; Daniels, S.; Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.; Jacobson, A.; Pappin, D. J. Mol. Cell Proteomics 2004, 3, 1154-1169.
PR049802+
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