弗氏2a志贺菌2457T株碱性蛋白质组图谱的建立

目的:建立弗氏2a志贺菌2457T株的碱性蛋白质组图谱。方法:首先采用双向电泳技术对弗氏2a志贺菌2457T株表达的全部碱性菌体蛋白及碱性膜蛋白进行分离,再通过基质辅助激光解析/电离串联飞行时间质谱进行鉴定。结果:共鉴定到46个蛋白点,对应于38种蛋白质。结论:首次完成了弗氏2a志贺菌2457T株的碱性蛋白质组图谱。     

小峰熊蜂蜂王蛹期发育蛋白质组分析

为了探明小峰熊蜂Bombus hypocrita蜂王蛹期发育蛋白质表达调控方面的特点,揭示其发育的分子机理。采用双向电泳法对小峰熊蜂蜂王蛹期发育进行蛋白质组研究,结果在小峰熊蜂蜂王蛹期的白眼期(A期)、褐眼期(B期)和黑眼期(C期)分别检测到81、80和75个蛋白点,特有蛋白质分别为8个、7个和2个,共有蛋白质为61个,A期到B期有4个蛋白质显著上调,5个显著下调,B期到C期有7个蛋白质显著上调,1个显著下调,A期到C期有10个蛋白质显著上调,有4个显著下调。此外,3个蛋白质是在A、B期表达C期关闭,6个蛋白质A、C期表达,B期关闭,5个蛋白质A期关闭,而B、C期表达。初步表明小峰熊蜂蜂王从蛹期发育到成蜂过程中,不仅需要一些保守蛋白质来调控,而且还需要一些特异蛋白质。     

Gene expression during gonadal differentiation in the rat: A two-dimensional gel electrophoresis investigation

Gonadal protein patterns were studied during development in the rat by two-dimensional micro-gel electrophoresis. Specific proteins were detected in both the male and the female sex at the morphologically indifferent state (two female- and one male-specific) and during differentiation. At the onset of gonadal differentiation (day 14) two additional sex-specific proteins were discovered in the male and two in the female. These proteins remained expressed during further development. One testicular protein was restricted to the cytosol of the tunica albuginea. The other one was absent from the tunica. In the female gonad, the two proteins were membrane-specific, one present in germ cells, the other in somatic cells. In the testis, one additional protein was discovered at postnatal day 1. Thus according to biochemical criteria there is no indifferent state of gonadal development. The testis and ovary express sex-specific genes both before and after the onset of gonadal differentiation.     

Proteome Analysis of Cerebrospinal Fluid in Amyotrophic Lateral Sclerosis (ALS)

Cerebrospinal fluid (CSF) is a promising source of biomarkers in amyotrophic lateral sclerosis (ALS). Using the two-dimensional difference in gel electrophoresis (2-D-DIGE), we compared CSF samples from patients with ALS (n = 14) with those from normal controls (n = 14). Protein spots that showed significant differences between patients and controls were selected for further analysis by MALDI-TOF mass spectrometry. For validation of identified spots western blot analysis and ELISA was performed. We identified 2 proteins that were upregulated and 3 proteins that were down-regulated in CSF in ALS. Of these, two proteins (Zn-alpha-2-glycoprotein and ceruloplasmin precursor protein) have not been reported in CSF of patients with ALS so far. In contrast, several other proteins (transferrin, alpha-1-antitrypsin precursor and beta-2-microglobulin) seem to be unspecifically affected in different neurological diseases and may therefore be of limited value as disease-related biochemical markers in ALS. Further evaluation of the candidate proteins identified here is necessary.     

A Comparative Genomics Analysis of Codon Reassignments Reveals a Link with Mitochondrial Proteome Size and a Mechanism of Genetic Code Change Via Suppressor tRNAs

Using a comparative genomics approach we demonstrate a negative correlation between the number of codon reassignments undergone by 222 mitochondrial genomes and the mitochondrial genome size, the number of mitochondrial ORFs, and the sizes of the large and small subunit mitochondrial rRNAs. In addition, we show that the TGA-to-tryptophan codon reassignment, which has occurred 11 times in mitochondrial genomes, is found in mitochondrial genomes smaller than those which have not undergone the reassignment. We therefore propose that mitochondrial codon reassignments occur in a wide range of phyla, particularly in Metazoa, due to a reduced “proteomic constraint” on the mitochondrial genetic code, compared to the nuclear genetic code. The reduced proteomic constraint reflects the small size of the mitochondrial-encoded proteome and allows codon reassignments to occur with less likelihood of lethality. In addition, we demonstrate a striking link between nonsense codon reassignments and the decoding properties of naturally occurring nonsense suppressor tRNAs. This suggests that natural preexisting nonsense suppression facilitated nonsense codon reassignments and constitutes a novel mechanism of genetic code change. These findings explain for the first time the identity of the stop codons and amino acids reassigned in mitochondrial and nuclear genomes. Nonsense suppressor tRNAs provided the raw material for nonsense codon reassignments, implying that the properties of the tRNA anticodon have dictated the identity of nonsense codon reassignments. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. [Reviewing Editor: Dr. Laura Landweber]     

Mapping endpoints of partial proteolysis fragments from regulatory subunit of type I cyclic AMP-dependent protein kinase

Methods for mapping endpoints of partial proteolysis fragments from regulatory subunit of type I cyclic AMP-dependent protein kinase are described with a view to using such data for fine-structure analysis of mutations and/or modifications affecting the protein's electrostatic charge. Peptides generated from [35S]methionine-labeled regulatory subunit were separated by high-resolution two-dimensional gel electrophoresis. Sites of papain cleavage in denatured regulatory subunit were deduced from the kinetics of the appearance, molecular weights, and relative isoelectric points of the fragments produced. These sites and sites of chymotrypsin digestion in the native protein were confirmed by studying peptide overlaps. Carboxy-terminal peptides were identified both by overlaps with cyclic AMP-binding chymotryptic fragments and by their preferential labeling during polysome runoff mediated by pactamycin, an inhibitor of protein initiation. Since peptides containing modifications or mutations that alter protein charge can be identified by shifts in first-dimension isoelectric focusing gel positions, knowledge of fragment endpoints will permit rapid mapping of sites of such alterations by two-dimensional gel analysis of partial proteolytic digests. Such a mapping procedure is inexpensive, can be applied to partially purified proteins or to proteins eluted from polyacrylamide gels, requires only nanogram amounts of the protein of interest, and does not require sequence data to determine relative positions of peptides. Therefore, it provides an attractive alternative to more classical peptide analysis for studying point mutations in cellular proteins of low abundance.     

蛋白质组研究新前沿：定量蛋白质组学

在过去几年里,蛋白质组研究取得了令人鼓舞的进展,2DE-MS途径的自动化,多维色谱整合串联质谱的使用,弥补了一些用双向凝胶电泳分离蛋白质的技术缺陷;从稳定同位素标记到ICAT战略的提出,为准确定量在细胞或组织中发挥重要调节功能的低丰度蛋白质提供了一个较为理想的方法。同时,蛋白质芯片技术的不断发展,也极大的丰富了定量蛋白质组学的研究。就定量蛋白质组学及其相关技术研究进展作一简要综述。     

蛋白质组学在细菌应激反应研究中的应用

当外部生存环境发生变化时,细菌会在短时间内发生应激反应。利用双向凝胶电泳技术结合生物质谱鉴定的方法对细菌蛋白表达谱变化进行研究,是细菌转录谱变化研究的深入和扩展,是细菌应激反应研究中的新热点。综述了蛋白质组学在细菌应激反应研究中的应用现状和存在问题。     

Identification of genes and proteins induced during the lag and early exponential phase of lager brewing yeasts

AIMS: The aim of the present study is to identify genes and proteins whose expression is induced in lager brewing yeast during the lag phase and early exponential growth. METHODS AND RESULTS: Two-dimensional gel electrophoresis was used to identify proteins induced during the lag and early exponential phase of lager brewing yeast in minimal medium. The identified, early-induced proteins were Ade17p, Eno2p, Ilv5gp, Sam1p, Rps21p and Ssa2p. For most of these proteins, the patterns of induction differed from those of the corresponding genes. However, the genes had similar early expression patterns in minimal medium as observed during lager brewing conditions. The expression of previously identified early-induced genes in Saccharomyces cerevisiae grown in minimal medium, ADO1, ALD6, ASC1, ERG4, GPP1, RPL25, SSB1 and YKL056C, was also early induced in lager yeast under brewing conditions. CONCLUSIONS: The results indicate that the above-mentioned genes in general are induced during the lag phase and early exponential growth in Saccharomyces yeasts. The processes in which these genes take part are likely to play an important role during growth initiation. SIGNIFICANCE AND IMPACT OF THE STUDY: Increased knowledge regarding the early growth phase of lager brewing yeast was obtained. Further, the universality of the identified expression patterns suggests new methodologies for optimization and control of growth initiation during brewing fermentations.     

Mapping of noninvasion TnphoA mutations on the Escherichia coli O18:K1:H7 chromosome

Abstract The most virulent newborn meningitis-associated Escherichia coli are of the serotype O18: K1: H7. We previously isolated a large number of E. coli O18:K1:H7 mutants resulting from transposon Tn phoA mutagenesis that fail to invade brain microvascular endothelial cells. We have now determined the locations of 45 independent insertions. Twelve were localized to the 98 min region, containing a 120 kb segment that is characteristic of E. coli O18:K1:H7. Another, the previously described insertion ibe -10::Tn phoA , was localized to the 87 min region, containing a 20 kb segment found in this E. coli . These noninvasion mutations may define new O18:K1:H7 pathogenicity islands carrying genes for penetration of the blood-brain barrier of newborn mammals.     

Analysis of S-adenosylmethionine and related sulfur metabolites in animal tissues

A high-performance liquid chromatographic method was devised that separates S-adenosylmethionine and related sulfur metabolites on a Radial-PAK SCX cation-exchange column using a four-step NH4COOH/(NH4)2SO4 elution gradient. This new procedure permits, in a single run of 60 min, the quantitative analysis of S-adenosylmethionine, S-adenosylhomocysteine (AdoHcy), 5'-deoxy-5'-methylthioadenosine, decarboxylated S-adenosylmethionine, decarboxylated AdoHcy, inosylhomocysteine, and other related metabolites. Furthermore, this method allows the detection in rat tissues of novel sulfur metabolites, S-inosylhomocysteine and decarboxylated AdoHcy. Perturbation of the levels of some of these metabolites could be detected in rat livers and spleens after the administration of 3-deazaadenosine, an inhibitor of AdoHcy hydrolase, but could not be detected in rat adrenal glands. It is notable that decarboxylated AdoHcy disappeared in the livers of rats treated with 3-deazaadenosine. HeLa cells incubated with [35S]methionine displayed the incorporation of the labeled sulfur into S-adenosylmethionine, AdoHcy, decarboxylated S-adenosylmethionine, S-inosylhomocysteine, and 5'-deoxy-5'-methylthioadenosine.     

Proteome analysis of the farnesol-induced stress response in Aspergillus nidulans--The role of a putative dehydrin

The isoprenoid alcohol farnesol represents a quorum-sensing molecule in pathogenic yeasts, but was also shown to inhibit the growth of many filamentous fungi. In order to gain a deeper insight into the antifungal activity of farnesol, we performed 2D-differential gel electrophoretic analysis (2D-DIGE) of Aspergillus nidulans exposed to farnesol. We observed an increased abundance of antioxidative enzymes and proteins involved in protein folding and the ubiquitin-mediated protein degradation. A striking finding was the strong up-regulation of a dehydrin-like protein (DlpA). Expression analyses suggested the involvement of DlpA in the cellular response to oxidative, osmotic and cold stress. In line with these data, we demonstrated that dlpA expression was regulated by the MAP kinase SakA/HogA. The generation of both a dlpA Tet(on) antisense RNA-producing A. nidulans strain (dlpA-inv) and a ΔdlpA deletion mutant indicated a role of DlpA in conidiation and stress resistance of dormant conidia against heat and ROS. Furthermore, the production of the secondary metabolite sterigmatocystin was absent in both strains dlpA-inv and ΔdlpA. Our results demonstrate the complexity of the farnesol-mediated stress response in A. nidulans and describe a farnesol-inducible dehydrin-like protein that contributes to the high tolerance of resting conidia against oxidative and heat stress.     

Analysis of the synaptic vesicle proteome using three gel-based protein separation techniques

Synaptic vesicles are key organelles in neurotransmission. Their functions are governed by a unique set of integral and peripherally associated proteins. To obtain a complete protein inventory, we immunoisolated synaptic vesicles from rat brain to high purity and performed a gel-based analysis of the synaptic vesicle proteome. Since the high hydrophobicity of integral membrane proteins hampers their resolution by gel electrophoretic techniques, we applied in parallel three different gel electrophoretic methods for protein separation prior to MS. Synaptic vesicle proteins were subjected to either 1-D SDS-PAGE along with nano-LC ESI-MS/MS or to the 2-D gel electrophoretic techniques benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE, and double SDS (dSDS)-PAGE in combination with MALDI-TOF-MS. We demonstrate that the combination of all three methods provides a comprehensive survey of the proteinaceous inventory of the synaptic vesicle membrane compartment. The identified synaptic vesicle proteins include transporters, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), synapsins, rab and rab-interacting proteins, additional guanine nucleotide triphosphate (GTP) binding proteins, cytoskeletal proteins, and proteins modulating synaptic vesicle exo- and endocytosis. In addition, we identified novel proteins of unknown function. Our results demonstrate that the parallel application of three different gel-based approaches in combination with mass spectrometry permits a comprehensive analysis of the synaptic vesicle proteome that is considerably more complex than previously anticipated.     

Proteomic analysis of the cyanobacterium Anabaena sp. strain PCC7120 with two-dimensional gel electrophoresis and amino-terminal sequencing

A protein-gene linkage map of the cyanobacterium Anabaena sp. strain PCC7120 was successfully constructed for 123 relatively abundant proteins. The total proteins extracted from the cell were resolved by two-dimensional electrophoresis, and the amino-terminal sequences of the protein spots were determined. By comparing the determined amino-terminal sequences with the entire genome sequence, the putative translation initiation sites of 87 genes were successfully assigned on the genome. The elucidated sequence features surrounding the translation initiation sites were as follows: (1) GTG and TTG in addition to the ATG were used as rare initiation codons; (2) the core sequences (GAGG, GGAG and AGGA) of the Shine-Dalgarno sequence were identified in the appropriate position preceding the 51 initiation sites (58.6%); (3) the nucleotides at the two regions, from -35 to -33, and from -19 to -17 (relative to the first nucleotide in the initiation codon) were preferentially adenines or thymines; (4) the nucleotides at the region from -14 to -8 were preferentially purines; (5) the nucleotide at position -1 was biased towards non-guanine (96.6%); (6) the nucleotide at the position +5 was preferentially cytosine (63.2%). It was evident that removal of the translation initiator methionine was dependent on the side-chain bulkiness of the penultimate amino acid residue. The predicted putative signal peptide sequences were also indicated. Besides confirming the existence of many predicted proteins, the data will serve as a starting point for the study of signals important in post-translational processing and nucleotide sequences important in the initiation of translation.     

Rice Proteome Database: A Step toward Functional Analysis of the Rice Genome

The technique of proteome analysis using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has the power to monitor global changes that occur in the protein complement of tissues and subcellular compartments. In this study, the proteins of rice were cataloged, a rice proteome database was constructed, and a functional characterization of some of the identified proteins was undertaken. Proteins extracted from various tissues and subcellular compartments in rice were separated by 2D-PAGE and an image analyzer was used to construct a display of the proteins. The Rice Proteome Database contains 23 reference maps based on 2D-PAGE of proteins from various rice tissues and subcellular compartments. These reference maps comprise 13129 identified proteins, and the amino acid sequences of 5092 proteins are entered in the database. Major proteins involved in growth or stress responses were identified using the proteome approach. Some of these proteins, including a β-tubulin, calreticulin, and ribulose-1,5-bisphosphate carboxylase/oxygenase activase in rice, have unexpected functions. The information obtained from the Rice Proteome Database will aid in cloning the genes for and predicting the function of unknown proteins.     

GMx33: a novel family of trans-Golgi proteins identified by proteomics

The known functions of the Golgi complex include the sorting, packaging, post-translational modification, and transport of secretory proteins, membrane proteins, and lipids. Other functions still remain elusive to cell biologists. With the goal of identifying novel Golgi proteins, a proteomics project was undertaken to map the major proteins of the organelle using two-dimensional gels, to identify the unknowns using tandem mass spectrometry, and to screen for Golgi residents using GFP-fusion constructs. Multiple unknowns were identified, and the initial characterization of one of these proteins is reported here. GMx33α is a member of a conserved family of cytosolic Golgi-associated proteins with no known homology to any known functional domain or protein. Biochemical analyses show that GMx33α differentially partitions into all phases of multiple detergent extractions, and two-dimensional immunoblots reveal that there are multiple differentially modified forms of GMx33α associated with the Golgi, several of which are phosphorylated. Evidence suggests that these post-translational modifications regulate its association with the Golgi. GMx33α was not found on Golgi budded vesicles, and immuno-electron microscopy co-localizes GMx33α to the trans -face on the same three cisternae as TGN38 in normal rat kidney cells. This work represents the preliminary characterization of a novel family of trans -Golgi-associated proteins.     

不同生长日龄罗汉果中糖基转苷蛋白凝胶电泳分析

采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法对不同日龄罗汉果内的主要糖基转苷蛋白进行分析,寻找罗汉果生长过程中皂苷形成的关键蛋白,为生物转化法生产罗汉果甜苷提供科学依据。得到一较清晰的罗汉果糖基转苷蛋白电泳图谱。据电泳图谱分析,初步得出罗汉果糖基转苷蛋白的内在变化规律。结果表明:罗汉果中存在多种糖基转苷蛋白,不同生长日龄罗汉果的糖基转苷蛋白也不一致,在几个关键时期糖基转苷蛋白变化特别明显。     

Changes in protein and mRNA populations during the senescence of carnation petals

Developmental changes in polypeptide and mRNA popultions in carnation ( Dianthus caryophyllus L. cv. White Sim) petals were investigated during the senescence of harvested flowers. Total proteins were extracted from flower petals at various stages of senescence and subjected to separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Analysis of the Coomassie blue stained gels revealed polypeptides with apparent molecular weights of 76, 62, 35.5 and 24 kDa which increased, while those with molecular weights of 70.5, 67.5, 46.5 and 31 kDa decreased during petal senescence. Changes in mRNA populations were investigated by translating poly (A)⁺RNA, isolated from carnation petals, in vitro using the rabbit reticulocyte lysate system. Polypeptides synthesized in vitro were separated by one- and two-dimensional gel electrophoresis and visualized by fluorography. Three classes of mRNA's were associated with the senescence of carnation petals. The majority of the mRNA's were constitutive at all stages of senescence. Another class of mRNA's increased with the climacteric rise in ethylene production, which accompanied the onset of senescence. Their translation products were 81, 58, 42, 38 and 35 kDa. In addition, several mRNA's appeared to decrease in abundance during the course of petal senescence. These results indicate that senescence of carnation flower petals is associated with changes in gene expression.