Proteomics analysis of cerebral cortex in Wistar rats

To analyze the protein expression pattern of the cerebral cortex in Wistar rats using the proteomics approach, proteins were separated by two-dimensional gel electrophoresis, stained with Coomassie brilliant blue and digested with trypsin. Then, we analyzed the peptide section using a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and identified the protein by indexing special database (SwissProt) according to the finger printing of the peptide quality. Eighty-four protein spots were identified, including metabolic enzymes, skeleton proteins, heat shock proteins, antioxidant proteins, signaling proteins, proteasome related proteins, neuron and glial specific proteins and serum associated proteins. The result of this study enriches the database of the proteome in the cerebral cortex of rats and lays a foundation for further research of neurological disorders in rat models. __________ Translated from Acta Biophysica Sinica, 2007, 23 (1): 151–156 [译自: 生物物理学报]     

大鼠脑皮质表达蛋白质组学研究

文章用蛋白质组学方法初步分析大鼠脑皮质蛋白质的表达。提取大鼠脑皮质蛋白质,双向凝胶电泳分离,考马斯亮蓝染色,胰蛋白酶胶内酶解,用基质辅助激光解吸/电离飞行时间质谱对酶解后的肽段进行分析,根据肽质量指纹图谱,检索专业数据库(Swissprot),对蛋白质进行鉴定。鉴定出84个蛋白,分别属于代谢酶、细胞骨架蛋白、热休克蛋白、抗氧化蛋白、信号传导蛋白、蛋白酶体相关蛋白、神经元特异蛋白及神经胶质蛋白等。文章结果丰富了大鼠脑皮质蛋白质组数据库,为在大鼠模型上研究神经疾病奠定了基础。     

正常与脑缺血大鼠的脑皮质蛋白质差异分析鉴定

Wistar大鼠随机分为正常组和模型组,采用改进的线栓法制备模型,在规定的时间点快速断头取脑,分离脑皮质组织,提取蛋白质后双向电泳展示,以ImageMaster 2D Elite v301软件对2_DE图谱进行差异表达分析,目标蛋白点用基质辅助激光解析电离质谱测定肽质量指纹图进行鉴定。线粒体应激70蛋白前体、血小板活化因子乙酰基水解酶Ibβ亚单位、ADP核糖基化因子蛋白3、电压依赖性阴离子选择通道蛋白1、泛素C末端水解酶同工酶L1、突触结合蛋白等11个蛋白在模型6h组表达上调,谷胱甘肽S-转移酶omega 1、 谷胱甘肽S-转移酶P、Cu-Zn超氧化物歧化酶、 ATP合酶D链、G蛋白β亚单位1、微管蛋白β链15、苹果酸脱氢酶等15个蛋白在模型6h组表达上调。胆绿素还原酶B、细胞因子A4前体为模型组新出现点,腺苷酸激酶同工酶1在模型组消失,Thiore doxin peroxidase 1在模型组分为2个点。以双向电泳技术得到分辨率较好的电泳图谱,并初步鉴定脑缺血后差异表达蛋白,为深入研究缺血性脑损伤病理机制奠定了基础。     

Proteome analysis of abundantly expressed proteins from unfed larvae of the cattle tick, Boophilus microplus

Protein expression in unfed larvae of the cattle tick, Boophilus microplus, was characterized using gel electrophoresis and mass spectrometry in an effort to assemble a database of proteins produced at this stage of development. Soluble and insoluble proteins were extracted and resolved by two-dimensional (2D) gel electrophoresis. Twenty abundantly expressed larval proteins were selected for peptide mass mapping and for peptide sequencing by matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) and quadrupole time-of-flight (Q-ToF) tandem mass spectrometry (MS), respectively. Only one protein, tropomyosin, was unequivocally identified from its peptide mass map. Ten proteins were assigned putative identities based on BLAST searching of heterologous databases with peptide sequences. These included a cytoskeletal protein (troponin I), multiple cuticular proteins, a glycine-rich salivary gland-associated protein and proteins with a presumed housekeeping role (arginine kinase, a high-mobility group protein and a small heat shock protein). Eight additional proteins were identified by searching translated open reading frames of a B. microplus EST database (unpublished): putative fatty-acid binding protein, thioredoxin, glycine-rich salivary gland protein and additional cuticular proteins. One remaining protein was not identifiable, suggesting it may be a novel molecule. The ongoing assembly of this database contributes to our understanding of proteins expressed by the tick and provides a resource that can be mined for molecules that play a role in tick-host interactions.     

Protein identification from two-dimensional gel electrophoresis analysis of Klebsiella pneumoniae by combined use of mass spectrometry data and raw genome sequences

Separation of proteins by two-dimensional gel electrophoresis (2-DE) coupled with identification of proteins through peptide mass fingerprinting (PMF) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is the widely used technique for proteomic analysis. This approach relies, however, on the presence of the proteins studied in public-accessible protein databases or the availability of annotated genome sequences of an organism. In this work, we investigated the reliability of using raw genome sequences for identifying proteins by PMF without the need of additional information such as amino acid sequences. The method is demonstrated for proteomic analysis of Klebsiella pneumoniae grown anaerobically on glycerol. For 197 spots excised from 2-DE gels and submitted for mass spectrometric analysis 164 spots were clearly identified as 122 individual proteins. 95% of the 164 spots can be successfully identified merely by using peptide mass fingerprints and a strain-specific protein database (ProtKpn) constructed from the raw genome sequences of K. pneumoniae. Cross-species protein searching in the public databases mainly resulted in the identification of 57% of the 66 high expressed protein spots in comparison to 97% by using the ProtKpn database. 10 dha regulon related proteins that are essential for the initial enzymatic steps of anaerobic glycerol metabolism were successfully identified using the ProtKpn database, whereas none of them could be identified by cross-species searching. In conclusion, the use of strain-specific protein database constructed from raw genome sequences makes it possible to reliably identify most of the proteins from 2-DE analysis simply through peptide mass fingerprinting.     

Selective Increase in S-100β Protein by Aging in Rat Cerebral Cortex

Changes in the concentrations of nervous tissue-related proteins and their isoproteins, such as S-100 proteins (S-100 alpha and S-100 beta), enolase isozymes (alpha-enolase and gamma-enolase), and GTP-binding proteins (Go alpha, Gi2 alpha, and beta-subunits), were determined in the CNS of male rats of various ages (from 2 to 30 months old) by means of enzyme immunoassay. The weights of brains and the concentrations of soluble proteins in the cerebral cortex, cerebellum, and brainstem were constant during the observation period. The concentration of S-100 beta protein, which is predominantly localized in glial cells, increased gradually in the cerebral cortex with age; levels in the 25-month-old rats increased to approximately 150% of the levels in the young (2-month-old) rats. However, the S-100 beta concentrations in the cerebellum and brainstem were relatively constant, showing similar values in rats 2-30 months old. Levels of other proteins, including both neuronal (gamma-enolase and Go alpha) and glial (alpha-enolase and S-100 alpha) marker proteins, did not change significantly with age in the cerebral cortex, cerebellum, and brainstem. These results suggest that there is a close relation between the age-dependent changes of the CNS function and S-100 beta protein levels in the cerebral cortex.     

脑出血与脑缺血动物模型脾淋巴细胞的蛋白质组学研究

目的：通过比较正常与脑出血及脑缺血模型大鼠脾淋巴细胞蛋白质表达的差异,初步探讨细胞免疫功能与脑血管病之间的关系。方法：将SD大鼠随机分为正常组、脑出血模型组（采用VII型胶原酶诱导脑出血）和局灶性脑缺血模型组（采用线栓法造成大脑中动脉阻塞）,分离大鼠脾淋巴细胞,提取总蛋白质后进行双向凝胶电泳,考马斯亮蓝染色,PDQUEST软件分析,对差异蛋白质点采用基质辅助激光解析电离质谱（MALDI-TOF-MS）技术进行鉴定并分析。结果：胶质细胞成熟因子 等9个蛋白在脑出血和脑缺血模型组表达上调,膜联蛋白III在脑出血和脑缺血模型组表达下调。结论：建立了分辨率高重复性较好的脑出血及局灶性脑缺血脾淋巴细胞总蛋白的双向凝胶电泳图谱,并鉴定一些与脑血管病脑损伤相关的差异表达蛋白质,为深入研究脑血管病细胞免疫功能改变与脑血管病之间的关系奠定了基础。     

天麻蛋白质的双向电泳和肽质量指纹谱分析与鉴定

采用双向聚丙烯酰胺凝胶电泳和质谱技术对天麻染菌球茎皮层和不染菌的新生球茎皮层进行了比较蛋白质组分析与鉴定。双向电泳后在分子量 1 2～97kD、等电点 3～ 1 0范围内 ,每块胶分离到约 90 0个蛋白质点。对新生球茎中表达量明显增加的 5个蛋白质点用基质辅助激光解吸 电离飞行时间质谱 (MALDI TOFMS)进行肽质量指纹谱的分析 ,并通过检索不同的数据库进行蛋白质鉴定与功能预测 ,初步认为第 4号蛋白点是一个与转录有关的RNA结合蛋白。同时本文在天麻蛋白质组样品制备、数据库检索策略以及蛋白质鉴定成功率等方面进行了探讨。     

The Postsynaptic Density: Constituent and Associated Proteins Characterized by Electrophoresis, Immunoblotting, and Peptide Sequencing

The proteins of the postsynaptic density (PSD) fraction of cerebral cortex were resolved by two-dimensional electrophoresis (2DE) and more than 30 proteins identified by characteristic 2DE mobility, immunoblotting with specific antibodies, and N-terminal and peptide sequencing. The PSD fraction is enriched for spectrin, actin, tublin and microtubule associated protein II, myosin, enzymes of glycolysis, creatine kinase, elongation factor 1 alpha, and receptor protein. The three neurofilament proteins are detected but a 58-kDa protein is prominent and is, by peptide sequencing, the bovine homolog of the recently cloned 66-kDa neurofilament protein; in contrast to the latter, however, it is enriched in cerebrum compared with spinal cord. A 68-kDa protein is identified as a member of the hsp70/BiP family of proteins. A protein, designated dynamin, indicating its putative role as a microtubule motor, is identified as a major protein, is found, however, greatly enriched in the particulate fraction, and is significantly denaturant and detergent insoluble. A protein designated N-ethylmaleimide-sensitive factor is also detected. Thus, two proteins implicated in vesicular transport are present in the PSD fraction. Seven polyclonal antibodies were produced to 2DE separated and electroeluted proteins of the PSD and were identified by peptide sequence analysis and 2DE profile as the hsp70/BiP homologous protein, the novel neurofilament protein synapsin IIa, pyruvate kinase, dynamin, aconitase and an unknown contaminating protein, and a 115-kDa protein that by subcellular fractionation and immunoblotting is a diagnostic PSD molecule. In addition, peptide sequences are obtained for four additional higher molecular weight proteins of the PSD that are not related at the level of primary structure to any known proteins.     

Proteome analysis of the leukocytes from the American alligator (Alligator mississippiensis) using mass spectrometry

Mass spectrometry was used in conjunction with gel electrophoresis and liquid chromatography, to determine peptide sequences from American alligator (Alligator mississippiensis) leukocytes and to identify similar proteins based on homology. The goal of the study was to generate an initial database of proteins related to the alligator immune system. We have adopted a typical proteomics approach for this study. Proteins from leukocyte extracts were separated using two-dimensional gel electrophoresis and the major bands were excised, digested and analyzed by on-line nano-LC MS/MS to generate peptide sequences. The sequences generated were used to identify proteins and characterize their functions. The protein identity and characterization of the protein function were based on matching two or more peptides to the same protein by searching against the NCBI database using MASCOT and Basic Local Alignment Search Tool (BLAST). For those proteins with only one peptide matching, the phylum of the matched protein was considered. Forty-three proteins were identified that exhibit sequence similarities to proteins from other vertebrates. Proteins related to the cytoskeletal system were the most abundant proteins identified. These proteins are known to regulate cell mobility and phagocytosis. Several other peptides were matched to proteins that potentially have immune-related function.     

Comparison of protein and peptide prefractionation methods for the shotgun proteomic analysis of Synechocystis sp. PCC 6803

Proteome analysis by gel-free "shotgun" proteomics relies on the simplification of a peptide mixture before it is analyzed in a mass spectrometer. While separation on a reverse-phase (RP) liquid chromatographic column is widely employed, a variety of other methods have been used to fractionate both proteins and peptides before this step. We compared six different protein and peptide fractionation workflows, using Synechocystis sp. PCC 6803, a useful model cyanobacterium for potential exploitation to improve its production of hydrogen and other secondary metabolites. Pre-digestion protein separation was performed by strip-based isoelectric focusing, one-dimensional polyacrylamide gel electrophoresis, or weak anion exchange chromatography, while pre-RP peptide separation was accomplished by isoelectric focusing (IEF) or strong cation exchange chromatography. Peptides were identified using electrospray ionization quadrupole time of flight-tandem mass spectrometry. Mass spectrometry (MS) and tandem mass spectra were analyzed using ProID software employing both a single organism database and the entire NCBI non-redundant database, and a total of 776 proteins were identified using a stringent set of selection criteria. Method comparisons were made on the basis of the results obtained (number and types of proteins identified), as well as ease of use and other practical aspects. IEF-IEF protein and peptide fractionation prior to RP gave the best overall performance.     

Proteome analysis of hairy root from Panax ginseng C.A. Meyer using peptide fingerprinting, internal sequencing and expressed sequence tag data

As an initial step to the comprehensive proteomic analysis of Panax ginseng C. A. Meyer, protein mixtures extracted from the cultured hairy root of Panax ginseng were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). The protein spots were analyzed and identified by peptide finger printing and internal amino acid sequencing by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and electrospray ionization quadrupole-time of flight mass spectrometry (ESI Q-TOF MS), respectively. More than 300 protein spots were detected on silver stained two-dimensional (2-D) gels using pH 3-10, 4-7, and 4.5-5.5 gradients. Major protein spots (159) were analyzed by peptide fingerprinting or de novo sequencing and the functions of 91 of these proteins were identified. Protein identification was achieved using the expressed sequence tag (EST) database from Panax ginseng and the protein database of plants like Arabidopsis thaliana and Oryza sativa. However, peptide mass fingerprinting by MALDI-TOF MS alone was insufficient for protein identification because of the lack of a genome database for Panax ginseng. Only 17 of the 159 protein spots were verified by peptide mass fingerprinting using MALDI-TOF MS whereas 87 out of 102 protein spots, which included 13 of the 17 proteins identified by MALDI-TOF MS, were identified by internal amino acid sequencing using tandem mass spectrometry analysis by ESI Q-TOF MS. When the internal amino acid sequences were used as identification markers, the identification rate exceeded 85.3%, suggesting that a combination of internal sequencing and EST data analysis was an efficient identification method for proteome analysis of plants having incomplete genome data like ginseng. The 2-D patterns of the main root and leaves of Panax ginseng differed from that of the cultured hairy root, suggesting that some proteins are exclusively expressed by different tissues for specific cellular functions. Proteome analysis will undoubtedly be helpful for understanding the physiology of Panax ginseng.     

In-depth proteomic profiling of the normal human kidney glomerulus using two-dimensional protein prefractionation in combination with liquid chromatography-tandem mass spectrometry

The kidney glomerulus plays a pivotal role in ultrafiltration of plasma into urine and also is the locus of kidney disease progressing to chronic renal failure. We have focused proteomic analysis on the glomerulus that is most proximal to the disease locus. In the present study, we aimed to provide a confident, in-depth profiling of the glomerulus proteome. The glomeruli were highly purified from the kidney cortex from a male, 68-year-old patient who underwent nephroureterectomy due to ureter carcinoma. The patient was normal in clinical examinations including serum creatinine and urea levels and liver function, and did not receive any chemotherapy and radiotherapy. The cortical tissue was histologically normal, and no significant deposition of immunoglobulins and complement C3 was observed. We employed a novel strategy of protein separation using 1D (SDS-PAGE) and 2D (solution-phase IEF in combination with SDS-PAGE) prefractionation prior to the shotgun analysis with LC-MS/MS. The protein prefractionation produced 90 fractions, and eventually provided a confident set of identified proteins consisting of 6686 unique proteins (3679 proteins with two or more peptide matches and 3007 proteins with one peptide match), representing 2966 distinct genes. All the identified proteins were annotated and classified in terms of molecular function and biological process, compiled into 1D and 2D protein arrays, consisting of 15 and 75 sections, corresponding to the protein fractions which were defined by MW and pI range, and deposited on a Web-based database (http://www.hkupp.org). The most remarkable feature of the glomerulus proteome was a high incidence of identification of cytoskeleton-related proteins, presumably reflecting the well-developed, cytoskeletal organization of glomerular cells related to their physiological functions.     

A Heuristic method for assigning a false-discovery rate for protein identifications from Mascot database search results

MS/MS and database searching has emerged as a valuable technology for rapidly analyzing protein expression, localization, and post-translational modifications. The probability-based search engine Mascot has found widespread use as a tool to correlate tandem mass spectra with peptides in a sequence database. Although the Mascot scoring algorithm provides a probability-based model for peptide identification, the independent peptide scores do not correlate with the significance of the proteins to which they match. Herein, we describe a heuristic method for organizing proteins identified at a specified false-discovery rate using Mascot-matched peptides. We call this method PROVALT, and it uses peptide matches from a random database to calculate false-discovery rates for protein identifications and reduces a complex list of peptide matches to a nonredundant list of homologous protein groups. This method was evaluated using Mascot-identified peptides from a Trypanosoma cruzi epimastigote whole-cell lysate, which was separated by multidimensional LC and analyzed by MS/MS. PROVALT was then compared with the two traditional methods of protein identification when using Mascot, the single peptide score and cumulative protein score methods, and was shown to be superior to both in regards to the number of proteins identified and the inclusion of lower scoring nonrandom peptide matches.     

Proteomic analysis of cerebral synaptosomes isolated from rat model of alzheimer's disease

Alzheimer's disease (AD) is a common and devastating disease and there is no readily available biomarker to aid diagnosis or monitor progression of it. To further understand the pathogenic mechanism of AD, proteomic approach was used to study the cerebral synaptosomes proteins of rats injected with Abeta1-40. Compared with the untreated samples, 14 proteins were found apparently altered through 2-dimensional gel electrophoresis. 12 of them were down-regulated and 2 were up-regulated. Three proteins including alpha-2-globin chain, peptidyl-prolycis-trans isomerase A (PPIaseA) and cofilin-1 protein were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and SWISS-PROT database query. Alpha-2-globin chain has not been shown to be associated with AD. PPIaseA and cofilin-1 protein are correlated with cell apoptosis and signaling. The altered proteins identified may help to understand the pathogenesis of AD.     

Uptake and effects of melatonin on the synthesis of proteins by the rat cerebral cortex

Slices of rat parietal cerebral cortex took up and retained [³H] melatonin up to a tissue concentration about 4-fold to that present in the incubation medium. This phenomenon was time-dependent, maxima being observed after 180 min-incubations Eighty to 93% of the radioactivity present in the cerebral cortex slices was chromatographically identified as melatonin. Even at the highest melatonin concentration that could be dissolved in the incubation media, a constant proportion of [³H] melatonin was bound to cortical slices, indicating that within this concentration range, melatonin binding is independent of its concentration. Melatonin effects on protein synthesis in the rat cerebral cortex were investigated by studying the incorporation of [³H] L-leucine into proteins in cerebral cortex of rats injected s.c. with 10 or 100 μg/day of melatonin for 5 to 10 days. Both treatments caused leucine incorporation into proteins to increase significantly by about 50 to 60%.     

Proteomic analysis of the neutrophil proteins of the tammar wallaby (Macropus eugenii)

A proteomic analysis of neutrophils from the tammar wallaby, Macropus eugenii, has been performed. Neutrophils were isolated from peripheral blood using density gradient centrifugation with Histopaque-1077, followed by treatment with ammonium chloride to lyse residual erythrocytes. Two-dimensional gel electrophoresis (2-DE) of lysed neutrophils was undertaken followed by in-gel trypsin digest and nanoliquid chromatography coupled tandem mass spectrometry (LC-MS) analysis and database searches. Seventy-seven proteins were isolated, 53 of which could be identified with high confidence as primarily of cytosolic origin. Protein identifications were only possible by matching identical peptide sequences within the NCBInr mammalian database with the Mascot search program. Sequence identities were only deemed acceptable if more than three peptides were identified, the precursor/protein ion tolerances were less than ± 0.25 Da and the total Mowse scores were greater than 100. The validity of this approach was tested using a scrambled database where no single identified peptide showed Mowse scores greater than 55. This is the first report of the neutrophil proteins of any marsupial and represents a first step in examining the identity of proteins involved in innate defence in this marsupial.     

Proteasomal Activity in Synaptosomes Obtained from the Cerebral Structures of Rats Subjected to Long-Lasting Immobilization Stress

We studied the proteasomal activity in synaptosomes obtained from tissues of the cerebral cortex, cerebellum, and hippocampus, as well as in the cytoplasm of cells of these brain structures, of rats subjected to long-lasting immobilization stress. It was demonstrated that the chymotrypsin-like activity of proteasomes in synaptosomes of the cerebral cortex and hippocampus of stressed animals was significantly higher (380 and 560%, respectively) as compared with that observed in control rats. The chymotrypsin-like and peptidylglutamyl peptide hydrolase activities of proteasomes in the cytoplasm of cortical cells under stress conditions also increased (210 and 180%, respectively). These data show that the activity of a multicatalytic proteolytic complex is sharply increased in synaptic terminals of cells of the cerebral cortex and hippocampus of stressed animals. The above complex plays a crucial role in the utilization of short-lived proteins whose molecules form receptors and ion channels; the amount of such proteins is especially great in synaptic terminals.     

A study of the spatial protein organization of the postsynaptic density isolated from porcine cerebral cortex and cerebellum

Postsynaptic density (PSD) is a protein supramolecule lying underneath the postsynaptic membrane of excitatory synapses and has been implicated to play important roles in synaptic structure and function in mammalian central nervous system. Here, PSDs were isolated from two distinct regions of porcine brain, cerebral cortex and cerebellum. SDS-PAGE and Western blotting analyses indicated that cerebral and cerebellar PSDs consisted of a similar set of proteins with noticeable differences in the abundance of various proteins between these samples. Subsequently, protein localization in these PSDs was analyzed by using the Nano-Depth-Tagging method. This method involved the use of three synthetic reagents, as agarose beads whose surface was covalently linked with a fluorescent, photoactivable, and cleavable chemical crosslinker by spacers of varied lengths. After its application was verified by using a synthetic complex consisting of four layers of different proteins, the Nano-Depth-Tagging method was used here to yield information concerning the depth distribution of various proteins in the PSD. The results indicated that in both cerebral and cerebellar PSDs, glutamate receptors, actin, and actin binding proteins resided in the peripheral regions within ～ 10 nm deep from the surface and that scaffold proteins, tubulin subunits, microtubule-binding proteins, and membrane cytoskeleton proteins found in mammalian erythrocytes resided in the interiors deeper than 10 nm from the surface in the PSD. Finally, by using the immunoabsorption method, binding partner proteins of two proteins residing in the interiors, PSD-95 and α-tubulin, and those of two proteins residing in the peripheral regions, elongation factor-1α and calcium, calmodulin-dependent protein kinase II α subunit, of cerebral and cerebellar PSDs were identified. Overall, the results indicate a striking similarity in protein organization between the PSDs isolated from porcine cerebral cortex and cerebellum. A model of the molecular structure of the PSD has also been proposed here.