Hypothermia Affects Translocation of Numerous Cytoplasmic Proteins Following Global Cerebral Ischemia Maria Teilum,*,† Morten Krogh,‡ Tadeusz Wieloch,† and Gustav Mattiasson† Lab for Experimental Brain Research, BMC A13, Lund University, Sweden, and Computational Biology and Biological Physics, Department of Theoretical Physics, Lund University, Sweden Received January 30, 2007
Using a decapitation ischemia model, we studied translocation of proteins to and from the cytosol in normothermic (NT) and hypothermic (HT) rat brains. 2D gel analysis identified 74 proteins whose cytosolic level changed significantly after 15 min of ischemia. HT preserved the cytosolic levels of several glycolytic enzymes, as well as many plasticity related proteins, otherwise decreased following NT ischemia. The levels of redox-related proteins was lower in HT than in NT. Our results indicate that translocation of proteins to and from the cytosol is an important issue during ischemia. Keywords: 2D gel • cortex • cytoskeleton • glycolysis • LC-MS/MS • MALDI • plasticity • redox-sensitive
Brain tissue requires a constant supply of oxygen and nutrients. In conditions such as stroke (focal ischemia) or cardiac arrest (global ischemia), the blood supply is severely reduced or completely abolished, and the stores of oxygen and nutrients are rapidly consumed. A complex and multifactorial cascade of molecular events called the ischemic cascade1,2 is initiated, which includes depolarization of cells, release of glutamate, loss of ion homeostasis, increases in intracellular calcium, disturbances in cell signaling cascades, mitochondrial dysfunction, oxidative stress, initiation of cell death pathways, and inflammation. The stress imposed on brain cells by these processes may lead to neuronal death and damage of brain tissue, with the concomitant local loss of brain function. Cardiac arrest is accompanied by a very high mortality, and survivors often have severe neurological disabilities. Stroke is the third leading cause of death and adult disability in the United States and Europe. Cerebral ischemia thus represents a major clinical problem. However, the only treatments that presently have been shown to reduce clinical brain damage following ischemia are cooling of the brain (hypothermia) and thrombolysis. Mild hypothermia (3-4 °C) has been evaluated in clinical trials3 and shows reduced sequelae following resuscitation from cardiac arrest and neonatal cerebral ischemia. The mechanisms behind hypothermic neuroprotection are not known in detail. During an energy crisis resulting from global cerebral ischemia, the excitatory synapses are flooded with glutamate due to failure of the energy dependent glutamate re-uptake pumps. This leads to Ca2+ flooding into the cells and activation of a wide variety of cell death promoting signals. Hypothermia reduces many of these effects of ischemia,
diminishing ischemia-induced cell death.4 Hypothermia can delay the onset of and strongly reduce the released amount of glutamate (100%) GABA and dopamine (60%)5,6 and improve the ion homeostasis, as well as reduce the release of glycine; see ref 7 for a recent review. Hypothermia furthermore reduces brain metabolism; however, the reduction due to a 4° decrease in temperature is merely 20%,8 which in itself cannot account for the observed dramatic tissue-protective effect. Hypothermia may also decrease the formation of reactive oxygen species (ROS) during the ischemic phase,9 as well as during the reperfusion phase,10 thus inhibiting several pathways that may lead to brain damage following ischemia. Another protective effect of hypothermia involves temperature-sensitive proteinprotein interactions. Two proteins known to be important in the ischemic cascade, Protein Kinase C-gamma (PKC-γ) and Calcium/Calmodulin dependent kinase II-alpha (CamKII-R), translocate from the cytoplasm to the membrane fraction following an ischemic episode.11-13 These proteins are thought to hyperphosphorylate glutamate receptors at the synaptic membrane,14,15 increasing their sensitivity toward glutamate, which may aggravate the ischemic injury. This translocation is prevented by mild hypothermia.16,17 Further, hypothermia affects short-term plasticity by reducing actin dynamics and preventing accumulation of erroneously polymerized actin molecules during ischemia,18 thereby affecting detrimental synaptic motility and transmission.18-20 Consequently, it is conceivable that translocation of other proteins than CamKIIR or PKCγ to or from the cytoplasm and/or altered polymerization dynamics of cytoskeletal constituents are also part of the complex molecular events underlying the neuroprotective effect of hypothermia.
* To whom correspondence should be addressed. Dr Maria Teilum, Lab for Experimental Brain Research, BMC A13, 22184 Lund, Sweden. E-mail,
[email protected]. † Lab for Experimental Brain Research. ‡ Department of Theoretical Physics.
Previous studies on cytoplasmic protein changes following ischemia or hypothermia in the CNS have been performed on a selected number of known target proteins. A recent study analyzed expression changes following hypoxia in neonatal rat using 2D gels,21 but no large-scale study including a high
Introduction
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Published on Web 05/31/2007
10.1021/pr070057l CCC: $37.00
2007 American Chemical Society
Translocation of Numerous Cytoplasmic Proteins
number of adult animals has been conducted previously. The purpose of the present study was to, for the first time, use an unbiased proteomics-based method to analyze global alterations in cytoplasmic proteins following global ischemia and to investigate whether hypothermia affects translocation of cytoplasmic proteins. To accomplish this, we used a decapitation ischemia model to mimic global ischemia without recirculation. Hypothermia or normothermia was instituted during a 15 min ischemia, and cytosolic proteins from cortex were analyzed using 2D gel electrophoresis. We show that the level of 74 unique proteins changes significantly during global ischemia and that hypothermia modulates these changes with potentially neuroprotective effect.
Experimental Procedures Ischemia. Animal procedures were approved by the Malmo¨/ Lund Ethical Committee for Animal Research (M224-03), and care was taken to minimize any suffering of the animals throughout the procedures. Adult male Wistar rats, 340-370 g, were briefly sedated with halothane and decapitated. The heads were placed in a rubber glove and submerged in 33 °C (“hypothermia” (HT), n ) 6) or 37 °C (“normothermia” (NT), n ) 6) water baths for 15 min. A temperature probe was inserted in the heads from the cerebellum and placed approximately in the middle of the brain during ischemia to allow the temperature to be measured. The brains were subsequently removed rapidly and snap frozen at -80 °C. Control brains (“control” (C), n ) 5) were removed from the heads immediately after decapitation. The brains were stored at -80 °C until use. The ischemic procedures were performed on two separate days, including three animals from each ischemia group on each day. Dissection of Tissue. Neocortex from both hemispheres was dissected at -17 °C and stored at -80 °C until all tissue was ready for homogenization. Homogenization Protocol. The applied homogenization protocol was adapted from previous studies22 to allow use of nondetergent buffers. Cortical tissue was homogenized in homogenization buffer (1:10 w/v) (0.32 mM sucrose, 2 mM EGTA, 10 mM Tris, pH 7.4) with Complete Protease Inhibitor Cocktail tablets (Roche, 1/50 mL) using a 10 mL Kontes Teflon Homogenizer, size 22. The homogenate was centrifuged in a Beckman Avanti centrifuge with F1010 rotor at 1000× g for 10 min at 4 °C to remove debris. The supernatant was transferred to a 100.2 rotor and centrifuged at 100 000× g for 1 h at 4 °C in a Beckman Optima Ultracentrifuge, yielding a crude membrane fraction and a cytosolic fraction. The cytosolic fraction was transferred to 1.5 mL eppendorf tubes and precipitated over night at -20 °C using three volumes of ice-cold acetone. The pellets were stored at -80 °C until use. Sample Processing for 2D Gels. The pellets were solubilized in 50 µL of rehydration buffer (8 M Urea, 4% CHAPS, 2% Pharmalyte pH 3-10NL (GE healthcare)), and the protein concentration was determined using a protein micro assay (BioRad) according to the manufacturers instructions. 2D Gels. Twenty-four centimeter Immobiline drystrips pH 3-10 nonlinear (NL) gradient strips (GE Healthcare) were rehydrated for 12 h with rehydration buffer containing 450 µg sample protein, 0.002% Bromphenolblue, and 2.08 mg/mL DTT. Two technical replicates were performed for each animal. Isoelectric focusing (step-n-hold program: 1 h at 500 V, 2 h at 4000 V, 8 h at 8000 V, 50 V until equilibration) was performed on an IPGphor (GE Healthcare). After isoelectric focusing, the
research articles strips were submerged in equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M Urea, 30% glycerol (v/v), 2% SDS (w/v), 0.002% Bromophenol blue (w/v)) two times for 15 min. For the first incubation, 10 mg/mL DTT was included, whereas 25 mg/mL iodoacetamide was included in the second incubation. Following equilibration, the strips were layered in agarose on top of a 12.5% SDS-PA gel cast in-house with one glass plate silanized, and electrophoresis was conducted for 15 h with a constant power of 1 W/gel or until the Bromphenolblue reached the bottom of the gel. A size marker (Dual color, BioRad) was loaded on each gel. Twelve gels were processed in parallel on an Ettan DALT II system (GE healthcare). The gels were stained using Gelcode Blue Stain Reagent (Pierce Biotechnology) according to the manufacturers instructions and stored on a glass plate at 4 °C submerged in water until scanning. The technical replicates were processed in parallel for isoelectric focusing and gel separation. Samples from the different treatment groups were mixed within a batch of 12 gels (denoted a cassette henceforth). The gels were scanned on an ImageScanner (calibrated using Kodak step tablet no. 2), using LabScan 5.0 (both GE healthcare) to capture the images, and analyzed using ImageMaster 2D platinum 5.0 (GE Healthcare). Initially, automatic spot detection was performed (Smooth ) 2, Area ) 50, Saliency ) 7), and undetected spots were subsequently added manually. Spots, which on one gel appeared as one large spot, but on other gels appeared as two separate spots, were manually split to yield two spots on all gels. A reference gel was created from all gels, thus including all detected spots on all gels. Auto-matching of the reference gel to the gels was performed initially and was followed by manual matching and grouping of spots for all gels. No normalization of the images was performed. Intensities of the matched spots were exported from ImageMaster 2D platinum 5.0 and subjected to statistical analysis. After transmission scanning, six of the gels were destained and restained with Ruthenium II Tris (bathophenantroline disulfonate) (RuBP). RuBP was synthesized as described by Rabilloud,23 and staining was performed as described by Lamanda.24 Following overnight incubation in 40% EtOH/10% HAc, the gels were scanned on a Typhoon 9400 fluorescent scanner using ImageQuant TL software (both GE healthcare). The images were exported to ImageMaster 2D platinum 5.0 and analyzed as described above for coomassie stained gels. For each gel, the number of spots detected with the two staining procedures were compared. As RuBP did not detect more spots than coomassie, the remaining gels were not stained with RuBP, and all further analysis was performed on images of coomassie stained gels. Data Analysis of Gels/Statistics. Statistic calculations were performed with base 2 logarithms of spot volumes. The gels were run in three cassettes, and the results from cassette 3 were systematically different from the two other cassettes. A correction for this was made by, for each spot, subtracting a constant from the logarithmic volumes of cassette 3 samples such that the mean of logarithmic volumes in cassette 3 was equal to the mean in cassette 1 and 2 combined. Estimates of mean values in the normothermic, hypothermic, and control groups, fold changes between pairs of groups, and standard deviations and p-values of these quantities were calculated using an algorithm combining the information from the detected spots with the number of missing spots. Missing spots contain information because a protein is more likely to be unobserved on a gel if it is lowly expressed. The Sammon plot25 Journal of Proteome Research • Vol. 6, No. 7, 2007 2823
research articles was performed using Euclidean distance in the logarithmic volumes. All calculations were performed in the statistical language R.26 Values are presented as means (SD. Spot Picking. On the basis of the identified statistical differences between the three groups, including spots with p-value < 0.1, ImageMaster 2D platinum 5.0 was used to generate a pick-list. The pick-list was exported to an Ettan Spot Handling Workstation (GE Healthcare), and automated spot excision, using a pickerhead with 2 mm diameter, was performed for a total of two gels both containing control samples. Automatic in-gel digestion using 20 µg/mL trypsin (Promega) was performed. For one of the gels, the generated peptides were dissolved in 2.5 µL matrix, and 2.0 µL were deposited directly on a MALDI target plate (MicroMass, 96 wells), whereas digested samples from the second gel were deposited in a 96well plate, dried, and redissolved in 10 µL 0.1% formic acid and analyzed by LC-MS/MS. A total of 288 spots were picked for MALDI and 206 spots were picked for LC-MS/MS. MALDI. MALDI spectra were acquired in data-dependent mode on a MALDI-TOF instrument (M@LDI LRHT, Waters). An external calibration was made using mass values from yeast alcohol dehydrogenase. Each spectrum represented up to 200 laser shots. LC-MS/MS. The samples were analyzed on a Q-Tof Ultima API coupled to a CapLC (both Waters). The auto sampler injected 6 µL of sample, and the peptides were trapped on a precolumn (“Capillary”, C18, 300 µm × 5 mm, 5 µm, 100 Å, LC-Packings) and separated on a reversed phase analytical column (“Atlantis”, C18, 75 µm × 150 mm, 3 µm, 100 Å, Waters). The flow through the column was 200 nL/min. Solvent A consisted of 2% acetonitrile and 98% water, containing 0.1% formic acid. Solvent B consisted of 90% acetonitrile and 10% water, containing 0.1% formic acid. The following HPLC method was used: 5% B for 3 min. From 5-60% B for 42 min, from 60-80% B for 5 min, hold at 80% B for 25 min, 80-5% B for 1 min and 5% B for 15 min. Total run time ) 90 min. The mass spectrometer analysis was made using data directed analysis (DDA), to allow picking of the most intense peaks for MS/MS analysis. The mass range m/z was from 400 to 1600 for MS and from 50 to 1800 for MS/MS. Only spectra from ions with charge state 2 and 3 were acquired. Analysis of Spectra. Raw data from MALDI spectra were converted to text files and analyzed using an automated version of the PIUMS software.27,28 Raw LC-MS/MS data were converted into peak lists using ProteinLynx Global Server 2.2 (Waters), imported into Proteios 1.1 (www.proteios.org), and exported as m/z data files. These were analyzed using Mascot and X!Tandem software, searching the International Protein Index (IPI) rat database 3.18 and using reverse sequences to correct for false positive hits. Search results were imported to Proteios 1.1 and a list of identified proteins with a false discovery rate of 0.1% was created. For both MALDI and LCMS/MS analyses, carbamidomethylation was set as a fixed modification, methionine oxidation as a variable, and one missed cleavage was allowed. Dynamic peptide tolerance of 50-200 ppm was used for MALDI, whereas 100 ppm was used for LC-MS/MS. Assignment of ID to Spots. Identities were assigned to the individual spots based either on MALDI, LC-MS/MS, or both, using the IPI database. In general, LC-MS/MS data were seen as superior, e.g. if MALDI identified two possible proteins, but LC-MS/MS only identified one; this identity was assigned to the spot. See Table 1 and supplementary table A for identities. 2824
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For Table 1, an average change in ratio has been calculated for each protein. The average ratio was calculated as the ratio between the total volume of all spots assigned to the same identity (SD. No p-value is calculated for these proteins; an asterisk (*) is assigned if all spots show the same type of regulation (up- or down-regulation) and if 50% of their individual p-values are below 0.05. Gene Ontology. The corresponding gene symbols (e.g., Aco2) were used to group the proteins according to the gene ontology terms. A web-based version of GO:TermFinder, searching the RGD database, was used to bin the proteins in fewer classes.29 On the basis of the generated GO classes (see below), three specific categories including proteins related to glycolysis, plasticity/cytoskeleton, or redox-sensitive proteins were generated. In Figure 3, the ratios for each node were plotted to illustrate possible common expression patterns. Values were normalized to obtain “control” equal to one. For proteins identified in several spots, an average (SD was calculated and plotted. Ratios, rather than log ratio changes, were used to allow protein level ) 0.
Results/Discussion The present study analyzes the effect of temperature on translocation of proteins to and from the cytosol in rat cerebral cortex after 15 min of global ischemia. We analyzed cytosolic proteins from 17 rats divided in three experimental groups (control (n ) 5), normothermia (37 °C, n ) 6) and hypothermia (33 °C, n ) 6)) by 2D gel electrophoresis (two technical replicates) in combination with statistics, which allowed identification of 74 proteins, whose level changed significantly. Within the studied time interval, changes in protein levels could be caused by translocation to and from the cytosol (e.g., to/ from membranes, organelles, or the cytoskeleton), or by proteolysis. As protein synthesis is inhibited during ischemia,30 increased levels of proteins in the cytosol were not due to de novo synthesis. We will start this section by presenting and discussing results from sample preparation, analysis, and identification, and will then move on to functional analysis and relevance to hypothermic neuroprotection of the identified proteins.
Sample Preparation, Analysis, and Identification Animal Model. A decapitation ischemia model was used to mimic global cerebral ischemia without recirculation. This model is very reproducible and allow for tight control of ischemia, temperature, and time, which will in turn reduce inter-sample variation and provide optimum conditions for proteomic analysis of the samples. The purpose was to study changes caused by translocation to and from the cytosol, rather than changes caused by proteolysis, transcription, or translation; this model was thus ideal, as it does not allow for any recirculation after ischemia. Fifteen minutes of decapitation ischemia is furthermore known to mimic the widely used twovessel occlusion global ischemia very well. The proteins already established to translocate out of the cytosol following ischemia are known to behave in similar ways in hippocampus and cortex. To minimize the use of animals, yet allowing a thorough analysis, cortical tissue was analyzed. Animals. The brain temperature at the end of the ischemic episode was 36.8 ( 0.3 °C for NT. For HT a brain temperature of 33.3 ( 0.2 °C was reached approximately 10 min after
Translocation of Numerous Cytoplasmic Proteins
research articles
Figure 1. Representative 2D gel with annotations for proteins in the three selected functional groups. The pI range was nonlinear and ranged from pH 3-10. Size-range was from approximately 15 to 100 kDa. Gel size 26 × 20 cm. Protein name abbreviations: Ywhae, 14-3-3 protein ; Aco2, Aconitate hydratase; Actb, Actin; Akr1b4, Aldose reductase; Eno1, R-enolase; Snca, R-synuclein; Coro1a, Coronin1A; Dpysl2, Dihydropyrimidinase-related protein 2; Blvrb_predicted, Flavin reductase; Aldoc, Fructose-bisphosphate aldolase C; Gstm3, Glutathione S-transferase µ3; Gapdh, Glyceraldehyde-3-phosphate dehydrogenase; Ldhb, L-lactate dehydrogenase B chain; Prdx2, Peroxiredoxin-2; Prdx5, Peroxiredoxin-5; Prdx6, Peroxiredoxin-6; Park7, Protein DJ-1; Pacsin1, Protein kinase C and casein kinase substrate in neurons protein 1; Pkm2, Pyruvate kinase; Gdi1, Rab GDP dissociation inhibitor R; Stmn1, Stathmin; Sod1, Superoxide dismutase 1; Txn1, Thioredoxin; Tpi1, Triosephosphate isomerase; Tuba, Tubulin R; Tubb, Tubulin β; Wdr1, WD repeat protein 1.
initiation of ischemia. For control animals, the brain temperature was 37.1 ( 0.1 °C. Cytosolic Proteins. Sample preparation yielded the same amount of protein per gram of cortical tissue for all groups (approximately 130 mg/g). The buffer used to extract the cytoplasmic proteins did not contain any detergents or denaturants, thus preserving possible protein complexes during processing. Large filaments, such as actin-filaments with associated proteins, will be excluded by the last step of highspeed centrifugation, suggesting that association of proteins to or from such filaments will be reflected as a decrease or increase in protein level in our analysis. 2D Gels. For all cassettes, the number of matched spots in controls was 322 ( 38, in hypothermic 340 ( 32, and in normothermic animals 272 ( 51. The spot pattern in the gels run in cassette one (n ) 12) and two (n ) 12) were very similar. As described above, a systematic error was introduced for cassette three, containing material from three normothermic, one hypothermic, and one control animal, and a correction (see above) was made in the statistical analysis. Normothermic animals yielded significantly fewer spots than control and hypothermic animals. Conclusions made from these observation should however be made with caution because the normothermic animals are over-represented in cassette three, which generally yielded fewer detected spots. As can be seen from Figure 2A, a similar number of increases and decreases
in protein levels occurred in normothermic ischemia, suggesting that unspecific protein degradation in the normothermic animals did not affect our results. When focused on a 3-10 NL range 2D gel, it is inevitable that some proteins will not be completely resolved on the gel. We chose to load 450 µg protein on each gel, as this was found to yield reproducible and distinct spot-patterns, allowing both quantification and identification. We tested two types of staining (Coomassie and RuBP) on the same gel and found that coomassie staining detected a similar number of spots as RuBP. Coomassie was therefore used throughout the experiment. Statistical analysis of the spot intensities in the 2D gels showed that the number of proteins with increased and decreased levels was similar in both hypothermic and normothermic samples. The overall changes in levels compared to control were larger for normothermic samples than for hypothermic samples (Figure 2A). Reproducibility of the two technical replicates was generally very good (see, for example, samples HT1 and NT1 on the Sammon plot (Figure 2B)). For a few of the samples, the reproducibility was less convincing (see for example sample NT3 on the Sammon plot). Further, the Sammon plot of all samples show that the hypothermic and control samples cluster together, indicating a higher degree of similarity between these two groups compared to normothermic samples. Taken together, our results indicate that hypothermia reduces the overall ischemia-induced changes in Journal of Proteome Research • Vol. 6, No. 7, 2007 2825
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Table 1. Details for Proteins in the Three Functional Groupsa IPI
protein
N
HT/C
NT/C
HT/NT
function
Glycolysis IPI00464815.7
Enolase, alpha
4 0.82 ( 0.08
0.67 ( 0.13 * 1.23 ( 0.24
IPI00231736.6
Fructose-bisphosphate aldolase C
2 1.42 ( 0.15 * 0.55 ( 0.11 * 2.57 ( 0.53 * Fructose 1,6-bisphosphate T Dihydroxyacetone phosphate + Glyceraldehyde 3-phosphate
IPI00555252.1
Glyceraldehyde-3-phosphate dehydrogenase
5 1.14 ( 0.15
0.34 ( 0.07 * 3.38 ( 0.71 * 2 Glyceraldehyde 3-phosphate T 1,3-Bisphosphoglycerate + NADH
IPI00231783.4
Lactate dehydrogenase B
1 0.94 ( 0.11
0.53 ( 0.09 * 1.76 ( 0.30 * Pyruvate + NADH T acid + NAD+
IPI00231929.6
Puruvat kinase
4 1.50 ( 0.19 * 0.74 ( 0.15
IPI00231767.4
Triosephosphate isomerase
5 1.10 ( 0.05
1.31 ( 0.07 * 0.84 ( 0.04 * Dihydroxyacetone phosphate T Glyceraldehyde 3-phosphate
IPI00325135.3
14-3-3
2 0.84 ( 0.30
1.50 ( 0.59
IPI00189819.1 IPI00563379.1
Actin, all isoforms
3 0.77 ( 0.11
2.22 ( 0.43 * 0.34 ( 0.07 * Major building block of the cytoskeleton
2.02 ( 0.36
2-Phosphoglycerate T Phosphoenolpyruvate
Phosphoenolpyruvate + ADP T Pyruvate + ATP
Plasticity 0.56 ( 0.09 * Involved in trafficiking of membrane proteins
IPI00388903.2
R-synuclein
1 1.50 ( 0.49 * 2.18 ( 0.77 * 0.69 ( 0.10 * Involved in synaptic plasticity
IPI00210071.1
Coronin 1A
1 1.07 ( 0.14
IPI00192034.1
Dihydropyrimidinaserelated protein 2
3 1.64 ( 0.19 * 0.88 ( 0.24
IPI00208245.1
Protein kinase C and casein kinase substrate in neurons protein 1
2 0.82 ( 0.20
0.33 ( 0.20 * 2.46 ( 1.57 * Connects actin cytoskelleton with dynamin-mediated vesicle fission
IPI00324986.1
Rab GDP dissociation inhibitor R
1 0.80 ( 0.34
2.75 ( 0.95 * 0.29 ( 0.11 * Suppresses hyperexcitability of the CA1 pyramidal neurons, involved in plasticity
IPI00231697.4
Stathmin
1 1.22 ( 0.09 * 1.84 ( 0.22 * 0.66 ( 0.08 * Prevents assembly and promoted disassembly of microtubules
IPI00197885.1 IPI00475639.2 IPI00400573.1 IPI00362160.1 IPI00197579.1 IPI00215349.3
0.51 ( 0.33 * 2.12 ( 1.39 * Involved in actin cytoskeleton remodelling 1.88 ( 0.48 * Regulates microtubule assembly
Tubulin, all isoforms in all spots
13 1.03 ( 0.46
0.63 ( 0.28
1.64 ( 0.14
WD repeat protein 1
3 1.35 ( 0.17
0.59 ( 0.09
2.29 ( 0.26 * Induces disassembly of actin filaments
IPI00421539.3
Aconitase
4 1.32 ( 0.15 * 0.76 ( 0.18
IPI00231737.4
Aldose reductase
1 0.96 ( 0.06
IPI00392676.3 IPI00230942.4
Flavin reductase Glutathione S-transferase Mu3
1 0.96 ( 0.07 1.82 ( 0.29 * 0.53 ( 0.08 * Attenuates ROS-mediated cell damage 1 0.64 ( 0.08 * 0.76 ( 0.09 * 0.83 ( 0.09 Catalyzes detoxification of electrophilic compounds, thus protecting against toxic by-products of oxidative damage
IPI00201561.2
Peroxiredoxin 2
1 1.07 ( 0.06
1.22 ( 0.08 * 0.88 ( 0.05 * Thioredoxin peroxidase, cytosolic
IPI00205745.1
Peroxiredoxin 5
1 1.17 ( 0.40
1.93 ( 0.70
0.61 ( 0.15 * Thioredoxin peroxidase, cytosolic, and mitochondrial
IPI00231260.4
Peroxiredoxin 6
1 1.07 ( 0.07
0.92 ( 0.05
1.16 ( 0.06 * GSH peroxidase and A2 phospholipase
IPI00212523.1
Protein DJ-1
1 1.12 ( 0.08
1.45 ( 0.13 * 0.78 ( 0.06 * Inactivates ROS by self-oxidation
IPI00231643.4
Superoxide dismutase 1
2 0.91 ( 0.04 * 1.22 ( 0.04 * 0.75 ( 0.04 * Catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide
IPI00231368.4
Thioredoxin
1 0.70 ( 0.18
Major building block of microtubuli
Redox-related 1.73 ( 0.40 * Catalyzes isomerization of citrate to isocitrate. Inactivated by superoxides
1.25 ( 0.09 * 0.76 ( 0.05 * Catalyzes reduction of glucose to sorbitol. Activation affected by cytosolic redox state
1.27 ( 0.33
0.55 ( 0.14 * Facilitate reduction of other proteins
a IPI, identity according to International Protein Index; N, number of spots assigned to protein; HT/C, hypothermia vs. control ( SD; NT/C normothermia/ control ( SD; HT/NT, hypothermia/normothermia ( SD. * assigned if all spots show the same type of regulation (up- or down-regulation) and if 50% of their individual p-values are below 0.05. Note that for actin and tubulin all isoforms were included in the same ratio. Details for individual spots are available in Table A, Supporting Information.
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Figure 2. Distribution of regulations and Sammon plot. (A) Relative level for all the 119 uniquely identified proteins, whose level changed with p-value < 0.05 compared to any of the other experimental groups. Black line, increased in normothermia; gray line, decreased in normothermia. Values were normalized to obtain “control” values equal to one. For proteins identified in several spots, an average ( SD was calculated and plotted. For this plot, log ratios were plotted to illustrate equal distribution of up-regulations and down-regulations more clearly. An equal number of increases and decreases were found for both hypothermia and normothermia indicating that general proteolysis does not affect the obtained results. The amplitude of changes is smaller for hypothermia, indicating that hypothermia during ischemia diminishes the changes inflicted by ischemia. (B) Sammon plot of values from all gels included in study. C1-C4, C6, control samples; HT1-6, hypothermic samples; NT1-6, normothermic samples. The two technical replicates are plotted with identical designations. The Sammon map is a projection of samples to two dimensions that attempts to preserve the Euclidean distance between samples in the space of log2 abundances of all 706 spots. The scales on the axes are shown to make it possible to see the original distances. A distance of 10 corresponds to an average protein fold change of 1.09. The control and hypothermia samples cluster together, indicating a higher degree of similarity between these groups than with normothermia. The good reproducibility of the analysis is demonstrated by the close positioning of the two technical replicates (e.g., NT5) and by the clustering of animals from each group.
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Figure 3. Relative level of proteins in the three functional groups. Values were normalized to obtain “control” values equal to one. Black line, increased in normothermia; gray line, decreased in normothermia. A general reduction in glycolytic enzymes is seen in normothermia; this is prevented by hypothermia. The amplitude of changes in plasticity-related proteins is larger in normothermic brains than in hypothermic brains. A lower level of redoxrelated proteins is found in hypothermia, possibly indicating that the ROS is lower in these brains.
cytoplasmic protein profile compared to normothermia, rendering a situation that is closer to control and cell survival. Identification. A picklist for spots to be identified was generated by selecting proteins that changed from control to either normo- or hypothermia (p < 0.1). Two-hundred eightyeight spots were picked for MALDI, and among those, 206 spots were subsequently picked for LC-MS/MS. A total of 204 spots were successfully identified, 119 of which contained only one protein and changed with p < 0.05 from control to either hypothermic or normothermic ischemia, or from hypothermic ischemia to normothermic ischemia (see Supplementary Table A). In the 119 spots included, 74 unique proteins were present. Table 1 contains data for three functional classes of proteins (see below). For proteins identified in several spots, data are Journal of Proteome Research • Vol. 6, No. 7, 2007 2827
research articles presented as a mean for all the spots (SD. Changes in proteins that were represented by more than one spot were considered significant if all spots displayed the same type of regulation, and g50% of the changes had p-values of
