蛋白质组学技术筛选与鉴定癫痫疾病的差异蛋白质

应用蛋白质组学双向凝胶电泳(Two-dimensional gel electrophoresis, 2DE)和质谱技术, 定量分析和鉴定了癫痫组(n=3)和正常组(n=3)脑组织的差异表达蛋白, 以从蛋白质水平上揭示癫痫病的发机制. 结果表明, 凝胶图谱可辨识2500~3000个蛋白点, 对21个显著差异表达蛋白点进行质谱鉴定和SwissProt数据库检索, 得到17个癫痫差异蛋白, 其中2个蛋白在癫痫组织中表达上调, 15个蛋白表达下调. 部分蛋白与癫痫的关系属首次报道. 这些蛋白与癫痫的发生发展相关, 可能成为癫痫的分子标志物和药物治疗的靶向蛋白.     

3D-ASL灌注成像和血清Hcy、NPY水平与癫痫患者病情及认知功能的相关性探究

选取我院108例癫痫发作患者作为研究组,同期82例健康体检者设为对照组,均接受3D-ASL灌注成像检查和血清同型半胱氨酸(Hcy)、神经肽(NPY)水平检测.结果发现,研究组患者平均脑血流量(CBF)和简易智能精神状态检查量表(MMSE)评分低于对照组,血清Hcy、NPY水平高于对照组(P＜0.05);癫痫发作患者CB...     

Analysis of blood plasma proteins in patients with Alzheimer's disease by two-dimensional electrophoresis, sequence homology and immunodetection

Blood plasma proteins of patients with Alzheimer's disease (AD; senile dementia) and non-AD-type dementia were resolved by two-dimensional electrophoresis and identified by migration position in the electrophoresis pattern, sequence homology, and immunodetection by using antibodies. For the control experiments, blood plasma proteins of a healthy young individual and non-dementia patients were examined in a manner similar to that of the plasma samples of AD patients. In the plasma sample of the healthy young individual, more than 350 spots of silver-stained proteins were observed and among these spots, 73 spots were identified. Blood plasma proteins of the AD and non-AD-type dementia patients were compared with those of the control and non-dementia patients. In the blood plasma samples of five AD patients, three patients had apolipoprotein E4, and another patient showed apolipoprotein L and complement factor H. For the AD-related proteins apolipoprotein E, tau-1, and presenilin 2, proteins were examined by immunostaining with antibodies, in both AD and non-AD patients. Among the three samples of non-AD-type dementia patients, one was distinguishable by amyloid A proteins, and the other by haptoglobin isoforms.     

Serological comparative proteomics analysis of mitochondrial autoantibody‐negative and ‐positive primary biliary cirrhosis

Here, we investigated the pathogenesis of primary biliary cirrhosis (PBC) by using 2D‐DIGE to analyze serological differences between anti‐mitochondrial antibody (AMA)‐positive and ‐negative PBC patients. The study comprised 30 patients with PBC; 20 AMA‐positive and ten AMA‐negative patients matched for age, sex , and pathological stage. A screening group (four AMA‐positive and four AMA‐negative patients) was used for 2D‐DIGE. Protein spots that were differently abundant between the two groups were identified via dye intensity and MS. Nine candidate proteins were identified from these spots. Western blotting was used to verify two of the identified proteins, serum amyloid P‐component (SAP) and vitronectin (VN). VN levels were significantly higher in the sera of AMA‐negative PBC patients (p < 0.01), whereas no significant difference was found between the two groups for SAP. To our knowledge, this is the first study to use serological comparative proteomics to explore differences between AMA‐positive and ‐negative PBC patients. VN levels were higher in AMA‐negative PBC patients, and this finding could be related to the more severe bile duct destruction observed in this group.     

人卵巢癌耐药细胞株的比较蛋白质组分析

本文采用高分辨二维凝胶电泳分离技术对人卵巢癌细胞株COC1及其耐药细胞株COC1/DDP中的蛋白质进行分离和差异表达分析, 应用基质辅助激光解吸电离-飞行时间质谱对酶解多肽进行测定[即测定蛋白质的肽质量指纹图(Peptide mass fingerprinting, PMF)], 并通过相应的数据库搜索来鉴定蛋白质. 为获得更准确的检索结果, 采用串联质谱技术对各肽段进行氨基酸测序, 并应用IPI-HUMAN数据库对上述检索结果进一步加以确认.      

Proteome analysis of mouse brain: two-dimensional electrophoresis profiles of tissue proteins during the course of aging

Mouse brain proteins were isolated from five regions (cerebellum, cerebral cortex, hippocampus, striatum, and cervical spinal cord) at five ages from the 10th week to the 24th month, and separated by two-dimensional gel electrophoresis (2-DE). 2-DE was carried out with an immobilized pH gradient bar in the first dimension, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Over one thousand protein spots were visualized by silver staining and quantified by image processing. In the analyses, 58 protein spots were distinguishable among the above five brain regions, and 17 proteins were shown to be varied in quantity in the course of aging. Partial amino-terminal sequences and/or internal sequences for a total of 301 protein spots were analyzed. One hundred and eighty proteins appeared to have blocked N-termini and 122 proteins were identified. Twenty-seven new proteins were identified by sequence homology search. A mouse brain proteome database was constructed, which consists of the 2-DE map images and the respective spot data files with 15 related references.     

人脑枕叶区衰老进程的比较蛋白质组学研究

分别从23岁、64岁、72岁、83岁以及94岁无神经性和精神性疾病史个体大脑皮层枕叶区取样．制备蛋白质样晶．进行双向凝胶电泳(2-DE)、考马斯亮蓝染色、凝胶扫描和Image Master 2D Elite软件分析，每张胶上平均可检测到1000个以上蛋白质点．通过软件半定量分析．进一步研究了衰老过程中枕叶蛋白质的差异表达，发现随年龄增长有7种蛋白质有一致的显著上升或下降趋势．应用质谱进行肽质量指纹图谱(PMF)和／或肽序列标签(PST)分析．数据库检索共鉴定了11种蛋白质．其中有5种具有一致的上调或下调性．包括神经元突触结构蛋白低分子量神经丝蛋白(Neurofilament triplet L protein．NF—L)、参与抗氧化反应的硫氧还原蛋白过氧化物酶(Peroxiredoxin)、三羧酸循环关键酶(顺)乌头酸水合酶(Aconitate hydratase)和糖代谢途径中的关键酶烯醇化酶2(Enolase 2)以及分子伴侣蛋白T复合物蛋白l(T-complex protein 1)．首次建立了正常人脑枕叶区的双向电泳蛋白质表达图谱．针对人脑枕叶区蛋白质在衰老过程中的差异表达进行了研究，并对差异表达蛋白在衰老进程中可能的生物学意义做了探讨．     

Assessing matrix assisted laser desorption/ ionization-time of flight-mass spectrometry as a means of rapid embryo protein identification in rice.

Rice embryo proteins were separated by two-dimensional gel electrophoresis (2-DE). A total of 105 spots were digested with trypsin and the resultant peptides were analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Raw mass spectra were fully-automatically processed and searched with selected monoisotopic masses against SWISS-PROT/TrEMBL and NCBInr databases. High quality mass spectra were obtained from 53 spots, of which 36 spots were identified including 29 not registered in databases. Fifty percent of the rice embryo proteins resolved in 2-DE could not be identified, indicating more efficient sample preparation techniques need to be developed in the future. At least four to five matching peptides were found to be essential for unambiguous identification of rice embryo proteins; peptide matching of less than four lead to ambiguous results. The suitability of peptide mass fingerprinting method as a means of rapid embryo protein identification in rice was discussed.     

Human bronchoalveolar lavage fluid protein two-dimensional database: study of interstitial lung diseases

Recently, we published an analytical two-dimensional electrophoresis (2-DE) protein map of human bronchoalveolar lavage fluid (BALF) using a pool of BALFs from various patients. In this report, the effect of lung disorders on the protein composition of the lung epithelial lining fluid was investigated by 2-DE of BALFs from individual patients with well-defined interstitial lung diseases: sarcoidosis, idiopathic pulmonary fibrosis (IPF) and hypersensitivity pneumonitis (HP), using improved experimental conditions. On these gels, about 600-1000 stained protein spots could be identified in a BALF sample containing 25 microg of protein, and our original human BALF protein database has, therefore, been considerably extended. Altogether, 429 protein spots corresponding to 66 different proteins (including isoforms, protein subunits and fragments) were identified by microsequence analysis and by matching with a human blood plasma 2-DE protein map available in the SWISS-2DPAGE database. A human 2-DE BALF database was established and is available on the World Wide Web (http://www.umh.ac.be/-biochim/proteomic.htm+ ++). The significance of the modifications observed between the different lung pathologies is discussed with the aim of understanding the mechanistic bases of lung disease pathogenesis and finding new potential lung markers of disorders.     

A two-dimensional gel database of human plasma proteins.

An updated two-dimensional electrophoretic map of human plasma proteins is presented, together with a complete listing of the individual protein spots, their locations, size and isoelectric points relative to internal charge standards. Forty-nine polypeptide species are identified, many consisting of multiple spots differing in glycosylation or sequence (e.g., immunoglobulins). A further series of 35 as yet uncharacterized proteins is indicated.     

Development of an integrated approach for evaluation of 2-D gel image analysis: impact of multiple proteins in single spots on comparative proteomics in conventional 2-D gel/MALDI workflow

With 2-D gel mapping, it is often observed that essentially identical proteins migrate to different positions in the gel, while some seemingly well-resolved protein spots consist of multiple proteins. These observations can undermine the validity of gel-based comparative proteomic studies. Through a comparison of protein identifications using direct MALDI-TOF/TOF and LC-ESI-MS/MS analyses of 2-D gel separated proteins from cauliflower florets, we have developed an integrated approach to improve the accuracy and reliability of comparative 2-D electrophoresis. From 46 spots of interest, we identified 51 proteins by MALDI-TOF/TOF analysis and 108 proteins by LC-ESI-MS/MS. The results indicate that 75% of the analyzed spots contained multiple proteins. A comparison of hit rank for protein identifications showed that 37 out of 43 spots identified by MALDI matched the top-ranked hit from the ESI-MS/MS. By using the exponentially modified protein abundance index (emPAI) to determine the abundance of the individual component proteins for the spots containing multiple proteins, we found that the top-hit proteins from 40 out of 43 spots identified by MALDI matched the most abundant proteins determined by LC-MS/MS. Furthermore, our 2-D-GeLC-MS/MS results show that the top-hit proteins in 44 identified spots contributed on average 81% of the spots' staining intensity. This is the first quantitative measurement of the average rate of false assignment for direct MALDI analysis of 2-D gel spots using a new integrated workflow (2-D gel imaging, "2-D GeLC-MS/MS", and emPAI analysis). Here, the new approach is proposed as an alternative to traditional gel-based quantitative proteomics studies.     

Proteome analysis of the Chlamydia pneumoniae elementary body

Chlamydia pneumoniae is an obligate intracellular human pathogen that causes acute and chronic respiratory tract diseases and that has been implicated as a possible risk factor in the development of atherosclerotic heart disease. C. pneumoniae cultivated in Hep-2 cells were 35S-labeled and infectious elementary bodies (EB) were purified. The EB proteins were separated by two-dimensional gel electrophoresis. Excised protein spots were in-gel digested with trypsin and peptides were concentrated on reverse-phase chromatographic beads for identification analysis by matrix-assisted laser desorption/ionization-mass spectrometry. In the pH range from 3-11, 263 C. pneumoniae protein spots encoded from 167 genes were identified. These genes constitute 15% of the genome. The identified proteins include 31 hypothetical proteins. It has recently been suggested that EB should be able to synthesize ATP. This view may be strengthened by the identification of several proteins involved in energy metabolism. Furthermore, proteins have been found which are involved in the type III secretion apparatus important for pathogenesis of intracellular bacteria. Proteome maps and a table of all identified proteins have been made available on the world wide web at www.gram.au.dk.     

Chronic activation of CREB and p90RSK in human epileptic hippocampus

Mesial temporal lobe epilepsy (MTLE) is associated with severe neuronal death and reactive gliosis in hippocampus. However, the molecular mechanisms underlying these pathological changes remain unanswered. ERK has been reported chronically activated in reactive glia of human epileptic hippocampus. In the present study, we investigated which of the downstream signaling molecules of ERK would be involved in MTLE. Western blot analysis demonstrated that CREB and p90RSK were strongly activated in MTLE patients. Increase in the active forms of CREB and p90RSK resulted not only from the increase in their phosphorylation levels but also from the increase in the protein levels. Activation of CREB and p90RSK was noted in the whole subfields of hippocampus with Ammon's horn sclerosis (AHS) representing a distinctive cellular distribution. However, the common major change was present in proliferating reactive astrocytes. In contrast, their activation was not significant in adjacent temporal lobes despite the presence of a number of astrocytes expressing high levels of GFAP. Our results demonstrate that chronic activation CREB and p90RSK in the epileptic hippocampus may be closely associated with the histopathological changes of AHS.     

Proteomic analysis of Helicobacter pylori cellular proteins fractionated by ammonium sulfate precipitation

Among 1590 ORFs in the Helicobacter pylori genome, >250 have been identified as authentic genes by proteomic analysis. Low-abundance proteins need to be enriched to a minimal amount for MALDI-TOF analysis and salt precipitation has generally been used for protein enrichment. Here, a whole-cell extract of H. pylori strain 26695 was subjected to protein fractionation with stepwise concentrations of ammonium sulfate and the proteins were displayed by 2-DE. The protein spots were quantified using PDQUEST software and identified by peptide fingerprinting. The 2-DE profiles and intensities of individual protein spots differed among the protein fractions. Out of the 98 identified proteins, 61 were found in the stepwise ammonium sulfate fractions but not in the whole-cell extract. Out of these, 37 proteins, including KdsA, were found exclusively in a single fraction. In contrast, GroEL, UreA, UreB, TrxA, NapA, and FldA were ubiquitously present in all fractions. Iron-containing proteins such as NapA, SodB, CeuE, and Pfr were found predominantly in the 100% saturated ammonium sulfate precipitate. Additionally, 29 proteins were newly identified in this study. These data will facilitate the preparation of significant H. pylori proteins, as well as provide information about low-abundance proteins.     

Two-dimensional electrophoretic and immunoblot analysis of cell surface proteins of spiral-shaped and coccoid forms of Helicobacter pylori

Cell surface proteins of the human gastric pathogen Helicobacter pylori extracted during different in vitro growth phases were analyzed by one- and two-dimensional gelelectrophoresis (1-DE and 2-DE) and by 2-DE immunoblot. Broth-cultured H. pylori cells were stained with an acridine-orange dye to monitor the morphological status of the organism. In 2-day-cultures, 96% of the bacterial cells were spiral-shaped and four days later a morphological switch to coccoid forms occurred. In 10-day cultures spiral-shaped forms were not found. By 1-DE, proteins with the molecular masses of 87 and 120 kDa were detected in the 2-day cultures that disappeared in cells of 12-day cultures. A protein corresponding in size to the heat shock protein (GroEl homolog, Hsp60) and a 62 kDa protein, the ureaseB-subunit, were identified in extracted proteins of 2-, 8-, and 12-day cultures. 2-DE revealed an increased number of silver-stained spots of 8-day cultures (in average 250 spots) compared with protein extracted from 2-day cells (in average 160 spots). 2-DE immunoblots performed with sera containing antibodies to major H. pylori proteins such as the A- and B-subunits of urease and the Hsp60 showed similar reactivity to surface proteins extracted from 2-, 8-, and 12-day cultures, suggesting that these proteins remain immunologically intact. Pooled sera from infected patients absorbed with spiral-shaped cells showed an almost total blocking of the antibody reactivity to extracted coccoid proteins in 2-DE immunoblot. Eighteen spots were still visible, but this reactivity probably represents a solid overexpression by the coccoid cells of Hsp60 and ureaseB proteins and is thus difficult to block.     

Changes of voltage-dependent anion-selective channel proteins VDAC1 and VDAC2 brain levels in patients with Alzheimer's disease and Down syndrome

Voltage-dependent anion-selective channel proteins (VDACs) are pore-forming proteins found in the other mitochondrial membrane of all eukaryotes and in brain postsynaptic membranes. VDACs regulate anion fluxes of a series of metabolites including ATP, thus regulating mitochondrial metabolic functions. We determined protein levels of VDACs in individual post-mortem brain regions of patients with Down Syndrome (DS) and Alzheimer's disease (AD) using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-mass spectroscopy (MALDI-MS). VDAC1 (SWISS-PROT accession number P21796) and VDAC2 (P45880) were unambiguously identified and quantified, but VDAC3 was not found. The spots representing VDAC1 were separated with different p/s (p/7.5, 8.5, and 10.0) probably caused by post-translational modifications as, e.g., phosphorylation. In DS cerebellum, total VDAC1 protein was elevated significantly whereas VDAC2 did not show any significant alterations. In AD brains, VDAC1 p/10.0 was significantly reduced in temporal, frontal, and occipital cortex with the p/7.5 form elevated in occipital cortex. Total VDAC1 was significantly decreased in frontal cortex and thalamus. VDAC2 was significantly elevated in temporal cortex only. The biological meaning of our results may be derangement of voltage-dependent anion-selective channel function and reflecting impaired glucose, energy, and intermediary metabolism as well as apoptotic mechanisms.     

Proteomic Analysis of Differentially Expressed Proteins of Arabidopsis thaliana Response to Specialist Herbivore Plutella xylostella

The changes of the proteome in Arabidopsis thaliana leaves were examined by specialist Plutella xylostella.Analysis of about 1100 protein spots on each 2DE gel revealed 38 differentially expressed protein spots in abun-dance of which 34 proteins were identified by MALDI-TOF/TOF MS.Among the insect feeding responsive proteins,a few proteins involved in carbon metabolism were identified including proteins associated with the Calvin cycle in the chloroplast and TCA cycle in the mitochondria,indicating carbon metabolism related proteins may play crucial roles in induced defense response in plants under insect infestation.The analysis elucidates the subcellular location of proteins demonstrates that about 50% of proteins are in the chloroplast,which shows the chloroplast has a key role in the insect feeding response for plant.Gene expression analysis of 10 different proteins by quantitative real-time PCR shows that four proteins of the mRNA level were correlated well with the protein level.This study further dissected the nature of insect infestation as a stress signal and some novel insect feeding responsive proteins identified may play an important role in induced defence machanism for plant.     

Proteome analysis of human liver tumor tissue by two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-mass spectrometry for identification of disease-related proteins

Hepatocellular carcinoma (HCC) is a common malignancy worldwide and is a leading cause of death. To contribute to the development and improvement of molecular markers for diagnostics and prognostics and of therapeutic targets for the disease, we have largely expanded the currently available human liver tissue maps and studied the differential expression of proteins in normal and cancer tissues. Reference two-dimensional electrophoresis (2-DE) maps of human liver tumor tissue include labeled 2-DE images for total homogenate and soluble fraction separated on pH 3-10 gels, and also images for soluble fraction separated on pH 4-7 and pH 6-9 gels for a more detailed map. Proteins were separated in the first dimension by isoelectric focusing on immobilized pH gradient (IPG) strips, and by 7.5-17.5% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels in the second dimension. Protein identification was done by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-time of flight-mass spectrometry (DE-MALDI-TOF-MS). In total, 212 protein spots (117 spots in pH 4-7 map and 95 spots in pH 6-9) corresponding to 127 different polypeptide chains were identified. In the next step, we analyzed the differential protein expression of liver tumor samples, to find out candidates for liver cancer-associated proteins. Matched pairs of tissues from 11 liver cancer patients were analyzed for their 2-DE profiles. Protein expression was comparatively analyzed by use of image analysis software. Proteins whose expression levels were different by more than three-fold in at least 30% (four) of the patients were further analyzed. Numbers of protein spots overexpressed or underexpressed in tumor tissues as compared with nontumorous regions were 9 and 28, respectively. Among these 37 spots, 1 overexpressed and 15 underexpressed spots, corresponding to 11 proteins, were identified. The physiological significance of the differential expressions is discussed.     

Expression patterns of antioxidant proteins in brains of patients with sporadic Creutzfeldt-Jacob disease

Using two-dimensional gel electrophoresis (2-DE) and Western blot analysis, we were able to identify and quantify six antioxidant proteins, peroxiredoxin (Prx) I, Prx II, Prx III, 1-Cys Prx, putative peroxisomal antioxidant enzyme (PLP), and mitochondrial Mn superoxide dismutase (Mn-SOD) in two individual brain regions, cerebellum and frontal cortex of patients with sporadic Creutzfeldt-Jacob (sCJD). Among six antioxidant proteins, 1-Cys Prx showed significant increase (P > 0.05) in sCJD frontal cortex whereas Prx I was decreased (P > 0.01). In cerebellum, levels of all antioxidant proteins studied were comparable to those of controls. Our findings provide evidence for the link between aberrant expression of antioxidant proteins, 1-Cys Prx and Prx I and CJD neuropathogenesis and we discuss the neuropathological meaning of these dysregulated antioxidant proteins in sCJD brain.