Comparative Proteome Analysis of Human Temporal Cortex Lobes by Two-Dimensional Electrophoresis and Identification of Selected Common Proteins

Human brain proteins were isolated from left and right temporal cortex lobes at the age of 73, 23, 84 years and separated by two-dimensional gel electrophoresis (2-DE). 2-DE was carried out with an immobilized pH gradient strip in the first dimension and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Over 800 polypeptide spots were resolved with a silver-staining protocol by computerized 2-D gel analsis. Seven of the polypeptide spots were evidently distinguishable between human left and right temporal lobes. Four of the polypeptide spots were larger and three were smaller in human right temporal lobe. One of these three protein spots that have descendent expression in human right temporal lobe was identified as carbonyl reductase (NADPH) 1 by MALDI-TOF MS. Thirty-three common spots were identified by ESI-MS/MALDI-TOF MS/Edman sequencing and a protein database search. These identified proteins include some important enzymes and regulating proteins.     

Comparative proteomics and correlated signaling network of rat hippocampus in the pilocarpine model of temporal lobe epilepsy

In temporal lobe epilepsy (TLE), the seizure origin typically involves the hippocampal formation. The pilocarpine-induced TLE provides a model to investigate the molecular and functional characterization of epileptogenesis by mimicking the human epileptic condition. Here, we employed a 2-D gel-based proteomic technique to profile proteome changes in the rat hippocampus after pilocarpine treatment. Using MALDI MS and MS/MS, 57 differentially expressed proteins were identified, which were found either up-regulated and/or down-regulated at the two time points 12 h (acute period; Ap) and 72 h (silent period; Sp) compared with the control. These proteins can be related to underlying mechanism of pilocarpine-induced TLE, indicating cytoskeleton modification, altered synaptic function, mitochondrial dysfunction, changed ion channel, and chaperone. Five of the identified proteins, synaptosomal-associated protein 25 (SNAP25), synapsin-2 (SYN2), homer protein homolog 2 (HOMER2), alpha-internexin (INA), and voltage-dependent anion channel 2 (VDAC2) were investigated by semiquantitative RT-PCR, and SNAP25 and INA were further validated by Western blot and immunohistochemistry staining. Furthermore, association of these pilocarpine-induced proteins with biological functions using the Ingenuity Pathway Analysis (IPA) tool showed that nucleic acid metabolism, system development, tissue and cell morphology were significantly altered. IPA of the canonical networks indicated that six membrane proteins (e.g., SNAP25, SYN2, and HOMER2) participated in three biological networks as starting proteins. Our results offer a clue to identify biomarkers for the development of pharmacological therapies targeted at epilepsy.     

Isoform- and subcellular fraction-specific differences in hippocampal 14-3-3 levels following experimentally evoked seizures and in human temporal lobe epilepsy

14-3-3 proteins are a family of signaling molecules involved in diverse cellular functions, which can mediate anti-apoptotic effects. Seizure-induced neuronal death may involve programmed (apoptotic) cell death pathways and is associated with a decline in brain 14-3-3 levels. Presently, we investigated the subcellular localization and effects of seizures on isoforms of 14-3-3 in rat hippocampus, and contrasted these to findings in human temporal lobe epilepsy (TLE). All brain isoforms of 14-3-3 were detected in the cytoplasmic compartment of rat hippocampus, while 14-3-3gamma and -zeta were also present in mitochondrial and microsome-enriched fractions. Focally evoked seizures in rats significantly reduced 14-3-3gamma levels within the microsome-enriched compartment at 4 h, with similar responses for 14-3-3zeta, while cytoplasm-localized 14-3-3beta, -epsilon and -eta remained unchanged. Analysis of human autopsy control hippocampus revealed similar 14-3-3 isoform expression profiles. In TLE samples, the microsome-enriched fraction also showed differences, but here 14-3-3epsilon and -zeta levels were higher than controls. TLE sample 14-3-3 isoform abundance within the cytoplasmic fraction was not different to controls. This study defines the subcellular localization of 14-3-3 isoforms in rat and human hippocampus and identifies the microsome-enriched fraction as the main site of altered 14-3-3 levels in response to acute prolonged and chronic recurrent seizures.     

Up-regulation of apelin in brain tissue of patients with epilepsy and an epileptic rat model

Prolonged epileptic seizures or SE can cause neuronal cell death. However, the exact role of neuroprotectant against brain injury during epileptic seizure needs to be further elucidated. The aim of this study was to investigate the expression of the apelin, a novel neuroprotective peptide, in brain tissues of the patients with temporal lobe epilepsy (TLE) and experimental rats using immunohistochemistry, immunofluorescence and Western blotting analysis and to discuss the possible role of apelin in TLE. Thirty temporal neocortical tissue samples from the patients with drug-refractory TLE underwent surgical therapy and nine histologically normal temporal lobes tissues as controls were used in our study. Fifty-six Sprague-Dawley rats were randomly divided into seven groups, including one control group and six groups with epilepsy induced by lithium-pilocarpine. Hippocampus and adjacent cortex were taken from the controls and epileptic rats at 1, 3, 7, 14, 30, and 60 days after onset of seizures. Apelin was mainly expressed in the neurons of TLE patients and controls, and was significantly increased in TLE patients compared with the controls. Apelin was also expressed in the neurons of experimental and control rats, it was gradually increased in the experimental rat post-seizure and reached a stable high level in chronic epileptic phase. Our results demonstrated that the increased expression of apelin in the brain may be involved in human TLE.     

Two-dimensional electrophoretic profiling of normal human kidney glomerulus proteome and construction of an extensible markup language (XML)-based database

To contribute to physiology and pathophysiology of the glomerulus of human kidney, we have launched a proteomic study of human glomerulus, and compiled a profile of proteins expressed in the glomerulus of normal human kidney by two-dimensional gel electrophoresis (2-DE) and identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and/or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Kidney cortices with normal appearance were obtained from patients under surgical nephrectomy due to renal tumor, and glomeruli were highly purified by a standard sieving method followed by picking-up under a phase-contrast microscope. The glomerular proteins were separated by 2-DE with 24 cm immobilized pH gradient strips in the 3-10 range in the first dimension and 26 x 20 cm sodium dodecyl sulfate polyacrylamide electrophoresis gels of 12.5% in the second dimension. Gels were silver-stained, and valid spots were processed for identification through an integrated robotic system that consisted of a spot picker, an in-gel digester, and a MALDI-TOF MS and / or a LC-MS/MS. From 2-DE gel images of glomeruli of four subjects with no apparent pathologic manifestations, a synthetic gel image of normal glomerular proteins was created. The synthetic gel image contained 1713 valid spots, of which 1559 spots were commonly observed in the respective 2-DE gels. Among the 1559 spots, 347 protein spots, representing 212 proteins, have so far been identified, and used for the construction of an extensible markup language (XML)-based database. The database is deposited on a web site (http://www.sw.nec.co.jp/bio/rd/hgldb/index.html) in a form accessible to researchers to contribute to proteomic studies of human glomerulus in health and disease.     

Identification of virulence factors and diagnostic markers using immunosecretome of Aspergillus fumigatus

Aspergillus fumigatus is a prime causative agent for various allergic and invasive aspergillosis. There has been a dramatic increase of such cases in last three decades yet the early diagnosis and virulence factor identification remains the challenge. In the present study secretome analysis of proteins isolated from the culture filtrate was done by 2D gel electrophoresis coupled with MS/MS and the immunosecretome analysis was carried out using immunoblotting of 2D transfer blots and probed with the sera of patients, immunized rabbit and mice. The identified proteins were analyzed further for homology with human proteins by BLAST search and for secretory signal by SignalP. A total of 65 protein spots from 2D gel resulted in identification of 24 different proteins along with their isoforms and out of which 15 proteins were identified as immunogenic in human. These findings may be helpful in the identification of virulence factors involved in aspergillosis and also useful as diagnostic markers.     

Increased Metallothionein I/II Expression in Patients with Temporal Lobe Epilepsy

In the central nervous system, zinc is released along with glutamate during neurotransmission and, in excess, can promote neuronal death. Experimental studies have shown that metallothioneins I/II (MT-I/II), which chelate free zinc, can affect seizures and reduce neuronal death after status epilepticus. Our aim was to evaluate the expression of MT-I/II in the hippocampus of patients with temporal lobe epilepsy (TLE). Hippocampi from patients with pharmacoresistant mesial temporal lobe epilepsy (MTLE) and patients with TLE associated with tumor or dysplasia (TLE-TD) were evaluated for expression of MT-I/II, for the vesicular zinc levels, and for neuronal, astroglial, and microglial populations. Compared to control cases, MTLE group displayed widespread increase in MT-I/II expression, astrogliosis, microgliosis and reduced neuronal population. In TLE-TD, the same changes were observed, except that were mainly confined to fascia dentata. Increased vesicular zinc was observed only in the inner molecular layer of MTLE patients, when compared to control cases. Correlation and linear regression analyses indicated an association between increased MT-I/II and increased astrogliosis in TLE. MT-I/II levels did not correlate with any clinical variables, but MTLE patients with secondary generalized seizures (SGS) had less MT-I/II than MTLE patients without SGS. In conclusion, MT-I/II expression was increased in hippocampi from TLE patients and our data suggest that it is associated with astrogliosis and may be associated with different seizure spread patterns.     

Proteome profiling of human epithelial ovarian cancer cell line TOV-112D

A proteome profiling of the epithelial ovarian cancer cell line TOV-112D was initiated as a protein expression reference in the study of ovarian cancer. Two complementary proteomic approaches were used in order to maximise protein identification: two-dimensional gel electrophoresis (2DE) protein separation coupled to matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and one-dimensional gel electrophoresis (1DE) coupled to liquid-chromatography tandem mass spectrometry (LC MS/MS). One hundred and seventy-two proteins have been identified among 288 spots selected on two-dimensional gels and a total of 579 proteins were identified with the 1DE LC MS/MS approach. This proteome profiling covers a wide range of protein expression and identifies several proteins known for their oncogenic properties. Bioinformatics tools were used to mine databases in order to determine whether the identified proteins have previously been implicated in pathways associated with carcinogenesis or cell proliferation. Indeed, several of the proteins have been reported to be specific ovarian cancer markers while others are common to many tumorigenic tissues or proliferating cells. The diversity of proteins found and their association with known oncogenic pathways validate this proteomic approach. The proteome 2D map of the TOV-112D cell line will provide a valuable resource in studies on differential protein expression of human ovarian carcinomas while the 1DE LC MS/MS approach gives a picture of the actual protein profile of the TOV-112D cell line. This work represents one of the most complete ovarian protein expression analysis reports to date and the first comparative study of gene expression profiling and proteomic patterns in ovarian cancer.     

Proteomic analysis of protein profiles during early development of the zebrafish, Danio rerio

In the present study, profiles of protein expression were examined during early development of zebrafish, an increasingly popular experimental model in vertebrate development and human diseases. By 2-DE, an initial increase in protein spots from 6 h post-fertilization (hpf) to 8-10 hpf was observed. There was no dramatic change in protein profiles up to 18 hpf, but significant changes occurred in subsequent stages. Interestingly, 49% of the proteins detected at 6 hpf remained detectable by 1 week of age. To map the protein expression patterns in 2-D gels, MALDI-TOF/TOF MS was employed to identify selected protein spots from early embryos. 108 protein spots were found to match known proteins and they were derived from 55 distinct genes. Interestingly, 11 (20%) of them produced multiple protein isoforms or distinct cleavage products. Although deyolked embryos were used in the analysis, a large number of vitellogenin derivatives remained prominently present in the embryos. Other than these, most of the identified proteins are cytosolic, cytoskeletal and nuclear proteins, which are involved in diversified functions such as metabolism, cytoskeleton, translation, protein degradation, etc. Some of the proteins with interesting temporal expression profiles during development are further discussed.     

Differential proteomics analysis of female and male adults of Angiostrongylus cantonensis

In this study, we identified the differentially expressed proteins of female and male adults of Angiostrongylus cantonensis through differential proteomics. We extracted and purified total proteins from male and female adults, separated proteins by two-dimensional difference gel electrophoresis (2D-DIGE) in pH 4-7, analyzed the gel images by DeCyder 7.0 software, and sacrificed the infected rats to count the number of male and female adults. It was found 28 protein spots that were differentially expressed; seven protein spots were then identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Five proteins were up-regulated and two proteins down-regulated in male adults compared with female adults. Three of the five up-regulated proteins with known functions ascribed to them were identified as galectin-1, proteasome alpha subunit and peroxiredoxin. The two down-regulated proteins were identified as indoleamine dioxygenase like-myoglobin and galectin. Furthermore, the female was significantly greater than male adults (P<0.01) in the rats. The findings demonstrate the differences in protein expression profiles and the ability to survive in the final host between female and male adults of A. cantonensis, and may provide a theoretical basis to study their developmental biology further.     

The effect of selenium enrichment on baker's yeast proteome

The use of regular yeast (RY) and selenium-enriched yeast (SEY) as dietary supplement is of interest because the Nutritional Prevention of Cancer (NPC) trial revealed that SEY but not RY decreased the incidence of prostate cancer (PC). Using two-dimensional difference in gel electrophoresis (2D-DIGE)-tandem mass spectrometry (MS/MS) approach, we performed proteomic analysis of RY and SEY to identify proteins that are differentially expressed as a result of selenium enrichment. 2D-DIGE revealed 96 candidate protein spots that were differentially expressed (p ≤ 0.05) between SEY and RY. The 96 spots were selected, sequenced by LC/MS/MS and 37 proteins were unequivocally identified. The 37 identified proteins were verified with ProteinProphet software and mapped to existing Gene Ontology categories. Furthermore, the expression profile of 5 of the proteins with validated or putative roles in the carcinogenesis process, and for which antibodies against human forms of the proteins are available commercially was verified by western analysis. This study provides evidence for the first time that SEY contains higher levels of Pyruvate Kinase, HSP70, and Elongation factor 2 and lower levels of Eukaryotic Translation Initiation Factor 5A-2 and Triosephosphate Isomerase than those found in RY.     

Proteomics in Drosophila melanogaster: first 2D database of larval hemolymph proteins

A proteomic approach was used for the identification of larval hemolymph proteins of Drosophila melanogaster. We report the initial establishment of a two-dimensional gel electrophoresis reference map for hemolymph proteins of third instar larvae of D. melanogaster. We used immobilized pH gradients of pH 4-7 (linear) and a 12-14% linear gradient polyacrylamide gel. The protein spots were silver-stained and analyzed by nanoLC-Q-Tof MS/MS (on-line nanoscale liquid chromatography quadrupole time of flight tandem mass spectrometry) or by Matrix assisted laser desorption time of flight MS (MALDI-TOF MS). Querying the SWISSPROT database with the mass spectrometric data yielded the identity of the proteins in the spots. The presented proteome map lists those protein spots identified to date. This map will be updated continuously and will serve as a reference database for investigators, studying changes at the protein level in different physiological conditions.     

Down-Regulation of CRMP-1 in Patients with Epilepsy and a Rat Model

The Collapsin Response Mediator Protein-1 (CRMP-1) is a brain specific protein identified as a signaling molecule of Semaphorin-3A and act as axon repellent guidance factor in nervous system. Recent studies indicated that axon guidance molecules may play a role in synaptic reorganization in the adult brain and thereby promote epileptogenesis. This study aimed to investigate expression pattern of CRMP-1 in epileptogenesis. Using double immunofluorescence labeling, immunohistochemistry and western blot analysis, we looked into the CRMP-1 expression in temporal neocortex from patients with temporal lobe epilepsy (TLE) and histological normal temporal neocortex from the controls. We also studied the expression pattern of CRMP-1 in hippocampus and adjacent cortex of a TLE rat model on 6, 24, 72 h, 1, 2 weeks, 1 month, and 2 months post-seizure, and from control rats. CRMP-1 was mainly expressed in the neuronal cytoplasm in the temporal lobe of intractable TLE patients, which was co-expressed with -2. CRMP-1 expression was downregulated in temporal neocortical of TLE patients. In addition, in pilocarpine-induced animal model of epilepsy, CRMP-1 dynamically decreased in a range of 2 months. Thus, our results indicate that CRMP-1 may be involved in the development of TLE.     

Comparison of membrane proteins from benign and malignant human thyroid tissues by two-dimensional polyacrylamide gel electrophoresis

In this study two-dimensional (2D) polyacrylamide gel electrophoresis with silver staining was used to analyze cellular membranous proteins of various normal and pathological human thyroid tissues. The aim was to understand the differences in cellular membranous proteins between these tissues, which would aid in the differential diagnosis of thyroid malignancy. Characteristic protein spots had a molecular mass of 50–64 kDa and a pI of 5.7-6.5. There were two groups of isoform protein spots in this area. The higher-molecular-mass group was found in follicular thyroid cancer tissues which and was not visible in normal thyroid tissues. The low-molecular-mass group was found in follicular carcinoma or adenoma tissues and was detected in one to three spots. The papillary thyroid carcinoma tissues gave different 2D gel maps. There were few spots of papillary thyroid carcinoma tissue membranous proteins within the examined area. The 2D gel maps may be used for differential diagnosis of follicular neoplasm. The characteristics of these protein spots require further investigation.