一株油藏嗜热厌氧杆菌的分离、鉴定及代谢产物特征

[目的]了解油藏环境中细菌的生理生化特性及代谢产物.[方法]采用Hungate厌氧操作技术从胜利油田罗801区块油层采出水中分离到一株厌氧杆菌SC-2.采用生理生化鉴定结合16S rDNA序列的系统发育学分析确定该菌株的系统发育地位,用气相色谱分析其代谢产物.[结果]菌株SC-2为严格厌氧的革兰氏阴性杆菌,菌体大小为0.38 um×1.7um-3.9um,单生、成对或成串生长,产端生芽孢.温度生长范围40℃-75℃(最适温度70℃);pH范围5.5-9.5(最适pH 6.5);NaCl浓度范围0%～5%(最适NaCl浓度0%).能够利用葡萄糖、麦芽糖、甘露糖、木糖等多种碳水化合物,发酵葡萄糖的产物是乙醇、乙酸、丙酸、H2、CO2及少量的乳酸.菌株SC-2的(G C)mol%含量为30.8%,与Thermoanaerobacter mathranii subsp.mathranii的16S rDNA序列相似性为99.85%.菌株利用葡萄糖产乙酸、乙醇的最佳初始pH为8.0;酵母粉能刺激生长并显著提高发酵葡萄糖的产酸、产醇率;培养基中添加4%(V/V)的乙醇能明显抑制菌体生长.[结论]菌株SC-2是从特殊生境(油层采出水)中分离到的一株嗜热、耐盐的厌氧菌,其发酵葡萄糖产生的代谢产物有利于改善油藏中的微环境.菌株SC-2与T.mathranii subsp.mathranii 11426T的最适pH和最大耐受NaCl浓度有所不同,且二者的(G C)mol%含量差异较大.     

Accurate quantitation of standard peptides used for quantitative proteomics

MS‐based proteomics has become an indispensable tool in system biology generating a need for accurate and precise quantitation of peptide standards. The presented method utilizes ultra performance LC‐MS/MS (UPLC‐MS/MS) to accurately quantify peptide standards at concentrations of 0.1–10 μM. The ability for accurate quantitation of micro‐molar concentrations has the advantages that quantitation can be performed routinely with high precision and the high sensitivity of the method minimizes the amounts required.     

Isolation and initial characterization of the mammalian midbody

Midbodies were isolated from synchronized cultures of Chinese hamster ovary (CHO) cells and their protein composition was studied by means of SDS PAGE. Gels of the midbodies included alpha and beta tubulins as major bands (approximately 30% of the total protein) and approximately 35 other bands, none of which constituted greater than 3.5% of the total protein. Extraction of the isolated midbodies with Sarkosyl NL-30- solubilized the midbody microtubules but left the central, dense matrix zone of the midbody intact. A protein doublet of approximately 115,000 mol wt was retained preferentially by the particulate fraction containing the matrix zones, indicating it to be a component of the matrix. The 115,000 mol wt doublet was also present in gels of isolated mitotic spindles from CHO cells. The overall protein composition of the isolated spindles was very similar to that of the isolated midbodies.     

Design and initial characterization of a circular permuted variant of the potent HIV-inactivating protein cyanovirin-N.

A circular permuted variant of the potent human immunodeficiency virus (HIV)-inactivating protein cyanovirin-N (CV-N) was constructed. New N- and C-termini were introduced into an exposed helical loop, and the original termini were linked using residues of the original loop. Since the three-dimensional structure of wild-type cyanovirin-N is a pseudodimer, the mutant essentially exhibits a swap between the two pseudo-symmetrically related halves. The expressed protein, which accumulates in the insoluble fraction, was purified, and conditions for in vitro refolding were established. During refolding, a transient dimeric species is also formed that converts to a monomer. Similar to the wild-type CV-N, the monomeric circular permuted protein exhibits reversible thermal unfolding and urea denaturation. The mutant is moderately less stable than the wild-type protein, but it displays significantly reduced anti-HIV activity. Using nuclear magnetic resonance spectroscopy, we demonstrate that this circular permuted monomeric molecule adopts the same fold as the wild-type protein. Characterization of these two architecturally very similar molecules allows us to embark, for the first time, on a structure guided focused mutational study, aimed at delineating crucial features for the extraordinary difference in the activity of these molecules.     

Further advances in the development of a data interchange standard for proteomics data

The Protein Standards Initiative (PSI) aims to define community standards for data representation in proteomics and to facilitate data comparison, exchange and verification. Significant progress was made in advancing the design and implementation of a draft standard for exchanging experimental data from proteomics experiments involving mass spectrometry at the 51st Annual Conference of the American Society for Mass Spectrometry. In collaboration with the American Society for Tests and Measurements, the PSI propose to publish this first draft at the forthcoming HUPO 2nd World Congress in Montreal, 8-11 October 2003.     

Immunopurification and initial characterization of dicotyledonous phytochrome

Antiserum was prepared against proteolytically undegraded phytochrome obtained from etiolated zucchini squash (Cucurbita pepo L., cv. Black Beauty). The antiserum was prepared by injecting into a rabbit immunoprecipitates between zucchini phytochrome and specific antiserum against undegraded oat (Avena sativa L., cv. Garry) phytochrome. Specific antiphytochrome immunoglobulins were purified from this crude serum by an affinity column consisting of conventionally purified undegraded pea phytochrome covalently linked to cyanogen bromide-activated agarose. These purified immunoglobulins were also linked to cyanogen bromide-activated agarose and were used to immunopurify zucchini, pea (Pisum sativum L., cv. Alaska), and lettuce (Lactuca sativa L., cv. Grand Rapids) phytochrome. All three dicotyledonous phytochromes exhibited a monomer size near 120,000 daltons by sodium dodecyl sulfate, polyacrylamide gel electrophoresis. Absorbance spectra of immunopurified zucchini phytochrome indicated that the ratio of visible to ultraviolet absorbance for purified zucchini phytochrome is lower than that observed for oat phytochrome. The isoelectric point of zucchini phytochrome, which was observed to be heterogeneous by this criterion, was found to be in the range of 6.5 to 7.0, higher than that observed for oat phytochrome. The electrophoretic mobility of zucchini phytochrome was found to be similar to that observed for oat and pea phytochrome under conditions that were nondenaturing and did not involve any molecular sieving effect. The amino acid analysis of zucchini phytochrome is similar to that reported previously for oat and rye (Secale cereale L., cv. Balbo) phytochrome.     

Use of lysozyme as a standard for evaluating the effectiveness of a proteomics process

Automated sequencing of unknowns in bottom-up proteomics makes the data produced susceptible to process control errors, which can be propagated into mistakes in analyte identification. Inclusion of an unintrusive internal standard, such as lysozyme, allows monitoring all phases of the proteomics process including sample preparation, enzymatic digestion, HPLC, mass spectrometry, and database searching. By using this internal standard, digestion issues including rearrangements, semi-tryptic fragments, and modifications were monitored. In addition, control of the HPLC process including column performance was achieved. The use of the lysozyme standard allowed easy optimization of mass spectral conditions including data dependent and collision induced dissociation settings. The use of this internal standard in a study of differential protein expression in rat serum samples is presented.     

Cloning and initial characterization of the Bordetella pertussis fur gene

In several Gram-negative pathogens the fur (ferric uptake regulator) gene product controls the expression of many genes involved in iron uptake and virulence. To facilitate the study of iron-regulated gene expression in Bordetella pertussis, we cloned the fur gene from this organism. The B. pertussis fur gene product was 54% identical to the Escherichia coli Fur and complemented two E. coli fur mutants. As with the E. coli fur gene, sequences upstream of the B. pertussis fur were homologous to the consensus Fur-binding site and to the consensus catabolite activator protein binding site.     

Isolation and initial characterization of aerobic chloromethane-utilizing bacteria

Abstract Eight strains of non-methane-utilizing aerobic methylotrophic bacteria able to grow on chloromethane as the carbon and energy source have been isolated. Based on their phenotypic and genomic characteristics the new isolates were classified as Hyphomicrobium spp. (strains CM1, CM2, CM9, CM29, CM35) and Methylobacterium spp. (strains CM4, CM30, CM34). All the strains possessed an inducible yet unknown enzyme that catalyzed conversion of chloromethane to HCl and formaldehyde. The latter was oxidized via formate to CO₂ or assimilated through icl⁺ or icl⁻ variants of the serine pathway.     

Shotgun proteomics for the characterization of subunit composition of mitochondrial complex I

Here we propose shotgun proteomics as an alternative method to gel-based bottom-up proteomic platform for the structural characterization of mitochondrial NADH:ubiquinone oxidoreductase (complex I). The approach is based on simultaneous identification of subunits after global digestion of the intact complex. Resulting mixture of tryptic peptides is purified, concentrated, separated and online analyzed using nano-scale reverse-phase nano-ESI-MS/MS in a single information dependent acquisition mode. The usefulness of the method is demonstrated in our work on the well described model system of complex I from bovine heart mitochondria. The shotgun method led to the identification and partial sequence characterization of 42 subunits representing more than 95% coverage of the complex. In particular, almost all nuclear (except MLRQ) and 5 mitochondria DNA encoded subunits (except ND4L and ND6) were identified. Furthermore, it was possible to identify 30 co-purified proteins of the inner mitochondrial membrane structurally not belonging to complex I. The method's efficiency is shown by comparing it to two classical 1 D gel-based strategies. Shotgun proteomics is less laborious, significantly faster and requires less sample material with minimal treatment, facilitating the screening for post-translational modifications and quantitative and qualitative differences of complex I subunits in genetic disorders.     

Methods of quantitative proteomics and their application to plant organelle characterization

Many cell biologists wish to know the subcellular localization of proteins of interest. Proteomics methods have the potential to describe the entire protein content of organelles. However, practical limitations in organelle isolation and analysis of low abundance proteins have meant that organelle proteomics has had, until recently, only limited success. Some examples of quantitative proteomic methods and their use in the study of plant organelle proteomes are discussed here. It is concluded that 2D-difference gel electrophoresis (2D-DIGE) as well as differential isotope tagging strategies coupled to non-gel-based LC-MS are proving useful in this area of research.     

Design and analysis issues in quantitative proteomics studies

Quantitative proteomics is the comparison of distinct proteomes which enables the identification of protein species which exhibit changes in expression or post-translational state in response to a given stimulus. Many different quantitative techniques are being utilized and generate large datasets. Independent of the technique used, these large datasets need robust data analysis to ensure valid conclusions are drawn from such studies. Approaches to address the problems that arise with large datasets are discussed to give insight into the types of statistical analyses of data appropriate for the various experimental strategies that can be employed by quantitative proteomic studies. This review also highlights the importance of employing a robust experimental design and highlights various issues surrounding the design of experiments. The concepts and examples discussed within will show how robust design and analysis will lead to confident results that will ensure quantitative proteomics delivers.     

Statistical validation of peptide identifications in large-scale proteomics using the target-decoy database search strategy and flexible mixture modeling

Reliable statistical validation of peptide and protein identifications is a top priority in large-scale mass spectrometry based proteomics. PeptideProphet is one of the computational tools commonly used for assessing the statistical confidence in peptide assignments to tandem mass spectra obtained using database search programs such as SEQUEST, MASCOT, or X! TANDEM. We present two flexible methods, the variable component mixture model and the semiparametric mixture model, that remove the restrictive parametric assumptions in the mixture modeling approach of PeptideProphet. Using a control protein mixture data set generated on an linear ion trap Fourier transform (LTQ-FT) mass spectrometer, we demonstrate that both methods improve parametric models in terms of the accuracy of probability estimates and the power to detect correct identifications controlling the false discovery rate to the same degree. The statistical approaches presented here require that the data set contain a sufficient number of decoy (known to be incorrect) peptide identifications, which can be obtained using the target-decoy database search strategy.     

Quantitative proteomics characterization on the antitumor effects of isodeoxyelephantopin against nasopharyngeal carcinoma

Isolated from Elephantopus scaber L., a Chinese medicinal herb that is widely used to prevent and treat cancers in China, isodeoxyelephantopin (ESI) exerted antitumor effects on several cancer cells. However, its antitumor mechanism is still not clear. In this study, we found that ESI could induce G2/M arrest and subsequently stimulate cell apoptosis in dose‐ and time‐dependent manners. We used SILAC quantitative proteomics to identify ESI‐regulated proteins in cancer cells, and found that 124 proteins were significantly altered in expression. Gene ontology and Ingenuity Pathway Analysis revealed that these proteins were mainly involved in the regulation of oxidative stress and inflammation response. Functional studies demonstrated that ESI induced G2/M arrest and apoptosis by inducing ROS generation, and that antioxidant N‐acetyl‐l ‐cysteine could block the ESI‐induced antitumor effects. Accumulated ROS resulted in DNA breakage, subsequent G2/M arrest and mitochondrial‐mediated apoptosis. ESI upregulated the expression of anticancer inflammation factors IL‐12a, IFN‐α, and IFN‐β through ROS‐dependent and independent pathways. The current work reveals that ESI exerts its antitumor effects through ROS‐dependent DNA damage, mitochondrial‐mediated apoptosis mechanism and antitumor inflammation factor pathway.     

牛精子蛋白质组学技术平台的建立及冻融前后精子差异蛋白初步分析

本研究通过探索不同的精子蛋白制备方法、水化液成分和优化2D电泳程序以建立牛精子蛋白质组学研究技术平台,同时以牛鲜冻精为实验材料通过差异凝胶电泳寻找冻融前后精子蛋白的改变。结果表明：使用改进的热TRIzol法裂解精子细胞制备蛋白,结合优化的2D电泳技术可建立稳定的牛精子蛋白质组学研究技术平台。差异凝胶电泳揭示牛精子在冻融后有质和量的改变：冻融后缺失的蛋白点有20个,表达下调的有2个,表达上调的有10个。作为一项阶段性的实验成果,本研究建立的2D平台和所发现的冻融引起的差异表达蛋白质点为揭示冷冻损伤机理和性控精液的差异蛋白质组学研究奠定了较好的基础。     

Fractionation and initial characterization of the kinetochore from mammalian metaphase chromosomes

We have partially isolated the kinetochore and associated centromeric structures from mammalian metaphase chromosomes. Human autoantibodies from scleroderma CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) patients were used as immunofluorescent probes to monitor fractionation. The procedure includes digestion of total chromosomal DNA with micrococcal nuclease, dehistonization with heparin, and dissociation of the remaining material with detergent and urea. We used a density gradient (metrizamide) to obtain an enriched fraction of stained material (kinetochore). When examined by electron microscopy, the kinetochore fraction is seen to contain numerous small immunoperoxidase-positive masses which are morphologically similar to the centromere/kinetochore region of intact metaphase chromosomes. The particulate fraction that contains kinetochore components represents less than 5% of total chromosomal proteins and contains less than 1% of total DNA. Two polypeptides of 18 and 80 kD were identified as kinetochore antigens by immunoblotting with CREST antiserum. In this paper we discuss the distribution of these kinetochore polypeptides with the associated centromeric chromatin.     

Isolation and initial characterization of the mouse Dnmt3l gene

We have isolated and sequenced the mouse zinc finger gene, Dnmt3l (DNA cytosine-5-methyltransferase 3-like), on mouse chromosome 10, showing similarity to members of the DNMT3/Dnmt3 family. The Dnmt3l protein contains an ADD zinc finger, which Dnmt3l shares with other Dnmt3 family members and Atrx. RT-PCR analysis showed Dnmt3l expression in testis, thymus, ovary, and heart, as well as in 7-day, 15-day, and 17-day mouse embryos.     

Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry

Post-translational modifications generate tremendous diversity, complexity and heterogeneity of gene products, and their determination is one of the main challenges in proteomics research. Recent developments in mass spectrometry based approaches for systematic, qualitative and quantitative determination of modified proteins promise to bring new insights on the dynamics and spatio-temporal control of protein activities by post-translational modifications, and reveal their roles in biological processes and pathogenic conditions. Combinations of affinity-based enrichment and extraction methods, multidimensional separation technologies and mass spectrometry are particularly attractive for systematic investigation of post-translationally modified proteins in proteomics.