OVNIp: An open source application facilitating the interpretation,the validation and the edition of proteomics data generated by MS analyses and de novo sequencing

Several academic software are available to help the validation and reporting of proteomics data generated by MS analyses. However, to our knowledge, none of them have been conceived to meet the particular needs generated by the study of organisms whose genomes are not sequenced. In that context, we have developed OVNIp, an open‐source application which facilitates the whole process of proteomics results interpretation. One of its unique attributes is its capacity to compile multiple results (from several search engines and/or several databank searches) with a resolution of conflicting interpretations. Moreover, OVNIp enables automated exploitation of de novo sequences generated from unassigned MS/MS spectra leading to higher sequence coverage and enhancing confidence in the identified proteins. The exploitation of these additional spectra might also identify novel proteins through a MS‐BLAST search, which can be easily ran from the OVNIp interface. Beyond this primary scope, OVNIp can also benefit to users who look for a simple standalone application to both visualize and confirm MS/MS result interpretations through a simple graphical interface and generate reports according to user‐defined forms which may integrate the prerequisites for publication. Sources, documentation and a stable release for Windows are available at http://wwwappli.nantes.inra.fr:8180/OVNIp .     

多环芳烃的真菌漆酶转化及污染土壤修复技术

漆酶可以转化多种有机污染物,在环境保护领域具有广泛的应用潜力。二十年来,通过多学科协同研究,对真菌漆酶转化多环芳烃的机制、特征等各方面的认识不断深入。基于漆酶等真菌木质素分解酶的污染土壤修复技术不断发展,并逐渐走向田间应用。本文首先介绍了真菌漆酶的一般作用机制与多环芳烃转化特征,结合我们的相关研究提出了漆酶作用下多环芳烃在土壤中的迁移模式;其次介绍了利用漆酶氧化原理修复污染农田土壤的潜力,着重对利用农业废弃物进行真菌生物刺激的修复实践进行了评述;最后,就漆酶转化多环芳烃基础研究中的若干重要问题进行了思考,并展望了真菌及其漆酶系统在污染土壤修复应用中的发展方向。     

Identification of plant mitochondrial proteins: A procedure linking two-dimensional gel electrophoresis to protein sequencing from PVDF membranes using a FastBlot cycle

Identification of the 329 spots visible in 2D gels of plant mitochondrial proteins is a challenge. This paper describes a 2D mini-gel protocol involving free-radical scavengers and purified reagents to make it compatible with protein sequencing, and evaluates its performance. The paper also describes a “FastBlot” sequencing cycle with the cycle time for protein sequencing from PVDF membranes reduced to less than 29 min with femtomole sensitivity. Other benefits of the cycle include reduced lag, reduced background, reduced loss of labile residues, and increased initial and repetitive yields. The procedure gave excellent results with maize mitochondrial proteins: of six protein spots that we tried to sequence, only one was blocked. The other spots yielded considerable sequence information. One spot was identified from the sequence as superoxide dismutase, while another spot corresponded to an unidentified cDNA from rice. The results of these experiments show that modifications of our previous procedures can provide good N-terminal protein sequencing from individual spots on 2D gels. The technique makes it possible to obtain sequence data, prepare gene probes, and identify many of the polypeptides in the 2D-gel map for plant mitochondria.     

Resolution ofFusarium sporotrichioides proteins by two-dimensional polyacrylamide gel electrophoresis and identification by sequence homology comparison in protein data base

Proteins fromFusarium sporotrichioides M-1-1, a T2-toxin-producing strain, were separated by two-dimensional polyacrylamide gel electrophoresis. One thousand two hundred and forty-four protein spots were resolved and 103 protein spots were subjected to N-terminal sequencing. Fifty-eight protein spots were sequenced and 48 proteins were observed to have blocked N termini. Forty out of 58 sequenced proteins were identified by homology search against the PIR protein sequence data base and protein superfamily data base, while the residual 18 sequences were not identified. Twenty-seven of the N-terminal-blocked proteins were subjected to mild anhydrous hydrazine vapor deblocking. Twenty-four spots were not deblocked indicating the presence of acyl groups at the N termini, while 3 proteins were deblocked showing the blocked group to be pyrroglutamyl carboxylic acid residues. The results can provide a more global view of cellular genetic expression than any other technique. The created data may offer a unique opportunity to link information with DNA sequence data.     

Determination of polycyclic aromatic hydrocarbons in urine of coke oven workers by headspace solid phase microextraction and gas chromatography-mass spectrometry

Polycyclic aromatic hydrocarbons (PAHs) represent a complex mixture of toxic compounds that are ubiquitous in the environment. We investigated the utility of head space-solid phase microextraction (HS-SPME) to measure the following surrogate PAHs in urine: naphthalene (NAP), phenanthrene (PHE), pyrene (PYR), and benzo(a)pyrene (BAP), representing classes of 2-, 3-, 4- and 5-ring compounds, respectively. We then applied the method to urine from 28 coke oven workers (median levels (microg/l) were: NAP=3.65, PHE=1.51, PYR=0.003, BAP not detected) and 22 controls (median (microg/l) NAP=0.859, PHE=0.062, PYR=0.001, BAP not detected). Urinary levels of NAP, PHE, and PYR were all associated with exposure category (controls, side- and bottom-workers, and top-workers) but not with smoking status. Strong correlations were observed between urinary levels of NAP, PHE, and PYR in coke-oven workers. Our results indicate that unmetabolized 2-, 3- and 4-ring PAHs can be measured in urine by HS-SPME. Such measurements can be used to investigate the uptake and metabolism of complex PAH mixtures in humans.     

Exploring membrane and cytoplasm proteomic responses of Alkalimonas amylolytica N10 to different external pHs with combination strategy of de novo peptide sequencing

Identification of differentially proteomic responses to external pHs would pave an access for understanding of survival mechanisms of bacteria living at extreme pH environment. We cultured Alkalimonas amylolytica N10 (N10), a novel alkaliphilic bacterium found in Lake Chahannor, in media with three different pHs and extracted the correspondent membrane and cytoplasm proteins for proteomic analysis through 2‐DE. The differential 2‐DE spots corresponding to the altered pHs were delivered to MALDI TOF/TOF MS for protein identification. Since the genomic data of strain N10 was unavailable, we encountered a problem at low rate of protein identification with 18.1%. We employed, therefore, a combined strategy of de novo sequencing to analyze MS/MS signals generated from MALDI TOF/TOF MS. A significantly improved rate of protein identification was thus achieved at over than 70.0%. Furthermore, we extensively investigated the expression of these pH‐dependent N10 genes using Western blot and real‐time PCR. The conclusions drawn from immunoblot and mRNA measurements were mostly in agreement with the proteomic observations. We conducted the bioinformatic analysis to all the pH‐dependent N10 proteins and found that some membrane proteins participated in iron transport were differentially expressed as external pH elevated and most of differential proteins with increased or bell‐shape mode of pH‐dependence were involved in bioenergetic process and metabolism of carbohydrates, fatty acid, amino acids, and nucleotides. Our data thus provide a functional profile of the pH‐responsive proteins in alkaliphiles, leading to elucidation of alkaliphilic‐adaptive mechanism.     

Sensitive biomarker of polycyclic aromatic hydrocarbons (PAHs): urinary 1-hydroxyprene glucuronide in relation to smoking and low ambient levels of exposure

The lysosomal neutral red retention time (NRRT) assay, a biomarker for lysosomal membrane stability, and the total immune activity (TIA) assay, a measure of non-specific immune system activity, were used in laboratory studies to assess the toxic effects of 2,4,6-trinitrotoluene (TNT) on earthworms (Eisenia andrei) in vivo. The results were compared with the concentration of TNT and its metabolites in earthworm tissue, as well as standard sublethal toxicity endpoints including growth (i.e. weight change) and reproduction effects from previously published studies. Filter paper experiments indicated a significant decrease in NRRT at ≥1.8 μg TNT cm^-2, whereas sublethal (weight loss) and lethal effects to earthworms were detected at ≥3.5 and 7.1 μg TNT cm^-2, respectively. Experiments in artificial soil showed that NRRT effects could be detected at lower TNT concentrations (≥55 mg TNT kg^-1 soil dry weight) compared with other sublethal endpoints (effects on growth and reproduction). The TIA biomarker did not significantly respond to TNT. Copper (as CuSO₄, filter paper contact tests) and 2-chloroacetamide (soil tests), which were used as reference toxicants, also decreased the NRRT. The use of the NRRT assay linked with tissue concentrations of TNT metabolites in earthworms was identified as a potentially appropriate biomarker approach for TNT exposure assessment under laboratory conditions and a novel tool for effects-based risk assessment.     

Identification of genes and proteins involved in the pleiotropic response to arsenic stress in Caenibacter arsenoxydans, a metalloresistant beta-proteobacterium with an unsequenced genome

The effect of high concentrations of arsenic has been investigated in Caenibacter arsenoxydans, a beta-proteobacterium isolated from an arsenic contaminated environment and able to oxidize arsenite to arsenate. As the genome of this bacterium has not yet been sequenced, the use of a specific proteomic approach based on nano-high performance liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) studies and de novo sequencing to perform cross-species protein identifications was necessary. In addition, a random mutational analysis was performed. Twenty-two proteins and 16 genes were shown to be differentially accumulated and expressed, respectively, in cells grown in the presence of arsenite. Two genes involved in arsenite oxidation and one in arsenite efflux as well as two proteins responsible for arsenate reduction were identified. Moreover, numerous genes and proteins belonging to various functional classes including information and regulation pathways, intermediary metabolism, cell envelope and cellular processes were also up- or down-regulated, which demonstrates that bacterial response to arsenic is pleiotropic.     

Identification of stool proteins in C57BL/6J mice by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry

Gastrointestinal disease is a major cause of mortality in humans and animals, and the detection of disease-associated protein in stool is an established diagnostic method in this context. Yet, no data currently exists about the protein composition of mammalian faeces. Using a newly developed two-dimensional (2D) gel method, 28 of the most abundant proteins in murine faeces were identified. Mammalian faeces contains protein from multiple species (from the individual, from gastrointestinal bacteria, from food, etc.). Yet, it was found that the majority of mouse stool proteins were of mouse origin, with a minority of proteins being derived from food (in particular soybean glycinin and conglycinin) and bacteria (flagellin). Most mouse proteins were proteases and saccharidases derived from the exocrine pancreas. In addition, two unexpected mouse proteins were identified: one was a newly described mucin-like protein from intestinal goblet cells (FcγBP); the other was the secreted form of carbonic anhydrase (type VI) from salivary gland. The data suggest that 2D analysis of faecal protein is likely to provide meaningful information about the physiological stage of the gastrointestinal tract. Compared with studies based on biopsies, faecal protein analysis may reduce the number of laboratory animals, and might also allow quicker bridging from animal studies to humans, where biopsy material is more difficult to obtain and is less relevant for general practice use.     

Preparation of genome-wide DNA fragment libraries using bisulfite in polyacrylamide gel electrophoresis slices with formamide denaturation and quality control for massively parallel sequencing by oligonucleotide ligation and detection

Bisulfite sequencing is widely used for analysis of DNA methylation status (i.e., 5-methylcytosine [5mC] vs. cytosine [C]) in CpG-rich or other loci in genomic DNA (gDNA). Such methods typically involve reaction of gDNA with bisulfite followed by polymerase chain reaction (PCR) amplification of specific regions of interest that, overall, converts C→T (thymine) and 5mC→C and then capillary sequencing to measure C versus T composition at CpG sites. Massively parallel sequencing by oligonucleotide ligation and detection (SOLiD) has recently enabled relatively low-cost whole genome sequencing, and it would be highly desirable to apply such massively parallel sequencing to bisulfite-converted whole genomes to determine DNA methylation status of an entire genome, which has heretofore not been reported. As an initial step toward achieving this goal, we have extended our ongoing interest in improving bisulfite conversion sample preparation to include a human genome-wide fragment library for SOliD. The current article features novel use of formamide denaturant during bisulfite conversion of a suitably constructed library directly in a band slice from polyacryamide gel electrophoresis (PAGE). To validate this new protocol for 5mC-protected fragment library conversion, which we refer to as Bis-PAGE, capillary-based size analysis and Sanger sequencing were carried out for individual amplicons derived from single-molecule PCR (smPCR) of randomly selected library fragments. smPCR/Capillary Sanger sequencing of approximately 200 amplicons unambiguously demonstrated greater than 99% C→T conversion. All of these approximately 200 Sanger sequences were analyzed with a previously published web-accessible bioinformatics tool (methBLAST) for mapping to human chromosomes, the results of which indicated random distribution of analyzed fragments across all chromosomes. Although these particular Bis-PAGE conversion and quality control methods were exemplified in the context of a fragment library for SOLiD, the concepts can be generalized to include other genome-wide library constructions intended for DNA methylation analysis by alternative high-throughput or massively parallelized methods that are currently available.     

Isoforms of a cuticular protein from larvae of the meal beetle, Tenebrio molitor, studied by mass spectrometry in combination with Edman degradation and two-dimensional polyacrylamide gel electrophoresis.

Simultaneous sequencing, using a combination of mass spectrometry and Edman degradation, of three approximately 15-kDa variants of a cuticular protein extracted from the meal beetle Tenebrio molitor larva is demonstrated. The information obtained by matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) time-course monitoring of enzymatic digests was found essential to identify the differences among the three variants and for alignment of the peptides in the sequence. To determine whether each individual insect larva contains all three protein variants, proteins extracted from single animals were separated by two-dimensional gel electrophoresis, electroeluted from the gel spots, and analyzed by MALDI MS. Molecular weights of the proteins present in each sample could be obtained, and mass spectrometric mapping of the peptides after digestion with trypsin gave additional information. The protein isoforms were found to be allelic variants.     

Two-dimensional gel analysis of secreted proteins induced by interleukin-1β in rat astrocytes

Interleukin-1β (IL-1β) is a pro-inflammatory cytokine produced in the brain by endogenous microglial cells responding to injury. Levels of IL-1β are elevated in several neurodegenerative disorders, including Alzheimer's disease. IL-1β, which can act as a mitogen for astrocytes, also elicits the expression and secretion of multiple factors and paracrine ‘second messengers’ such as other cytokines, nerve growth factor, prostaglandins and nitric oxide that may in turn modulate neuronal and glial responses to injury. Utilizing giant, high-resolution two-dimensional gel electrophoresis, we have sought to more fully define the potential range of protein mediators that are secreted by astrocytes treated with IL-1β. In cultured rat astrocytes, we observe dramatic increases in the secretion of eight different protein species after 24 h of treatment with human recombinant IL-1β (1 U/ml). Seven of the proteins are also induced by tumor necrosis factor-α or basic fibroblast growth factor. Based on immunoprecipitation with specific antisera, we have identified three of these proteins as plasminogen activator inhibitor type-1, ceruloplasmin, and complement component C3. The identities of the other proteins, including the IL-1β-specific induction, are currently unknown. Characterization of these downstream modulators of IL-1β action complements gene-based approaches and will provide a better understanding of astrocyte responses to injury as well as markers for astrocyte activation in neurodegenerative diseases.     

Detection of hypoxia-related proteins in medaka (Oryzias latipes) brain tissue by difference gel electrophoresis and de novo sequencing of 4-sulfophenyl isothiocyanate-derivatized peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Two-dimensional fluorescence-based difference gel electrophoresis (DIGE) was used in combination with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) to identify a set of hypoxia-related biomarker proteins in medaka (Oryzias latipes) brain tissue. Each of the proteins were identified via de novo sequencing of tryptic peptides derivatized with 4-sulfophenyl isothiocyanate (SPITC), which N-terminally sulfonates peptides and promotes facile post-source decay peptide fragmentation, resulting in greatly simplified spectra consisting mainly of y-series fragment ions. We also report that addition of the non-ionic surfactant n-octyl-beta-d-glucopyranoside significantly improves SPITC-derivatized peptide recoveries. In addition, we found that a MALDI matrix consisting of the sodium-tolerant matrix 2,4,6-trihydroxyacetophenone, diammonium citrate, and alpha-cyano-4-hydroxycinnamic acid also improves ionization of SPITC-peptides, presumably by reducing ionization suppression effects from matrix contaminants, especially sodium cations. The DIGE experiments and analyses resulted in detection of six abundant proteins and related isozymes up-regulated (>1.49, p<0.005) in hypoxic medaka brain tissues, including two hemoglobin beta subunit forms, four carbonic anhydrase 2 forms, calbindin, aldolase, succinate dehydrogenase, and glutathione-S-transferase.     

Identification of proteins adducted by reactive metabolites of naphthalene and 1-nitronaphthalene in dissected airways of rhesus macaques

Naphthalene and 1-nitronaphthalene are ambient air pollutants, which undergo P450-dependent bioactivation in the lung. Reactive metabolites of naphthalene and 1-nitronaphthalene covalently bind to proteins, and the formation of covalent adducts correlates with airway epithelial cell injury in rodent models. These studies were designed to identify protein adducts generated from these reactive metabolites within distal respiratory airways. Distal bronchioles and parenchyma from rhesus monkeys were incubated with [(14)C]naphthalene or [(14)C]1-nitronaphthalene. Proteins were separated by 2-DE, blotted to PVDF membranes, and adducted proteins imaged by storage phosphor analysis. MS of in-gel tryptic digests identified numerous adducted proteins including: eight cytoskeletal proteins, two chaperone proteins, seven metabolic enzymes, one redox protein, two proteins involved in ion balance and cell signaling, and two extracellular proteins. While many proteins are adducted by both naphthalene and 1-nitronaphthalene, some are unique to the individual toxicant and airway subcompartment. Although the role which adduction of these proteins plays in cytotoxicity was not evaluated, these studies provide candidate proteins for future work designed to determine the importance of protein adducts in the mechanisms of toxicity and for developing biomarkers useful in determining the relevance of findings in animal models to exposed human populations.     

Optimization and modeling of phenanthrene degradation by <Emphasis Type="Italic">Mycobacterium</Emphasis> sp. 6PY1 in a biphasic medium using response-surface methodology

In the present paper, the degradation of phenanthrene, a model polycyclic aromatic hydrocarbon compound, by the Mycobacterium strain 6PY1 was optimized in a biphasic culture medium. The optimization and modeling were performed using the design of experiments methodology. The temperature, the silicone oil/mineral salts medium volume ratio, and the initial cell concentration, were used as the central composite design parameters. In all experiments, the phenanthrene was degraded to undetectable levels. Response surface methodology was successfully employed to derive an empirical model describing the rate and time of degradation and to deduce the optimal degradation conditions. As a result of the optimization processes, the optimal responses for the degradation rate, the volumetric degradation rate, and the 90% degradation time were estimated to be 0.172 mg h⁻¹, 22 mg l⁻¹ h⁻¹, and 18 h, respectively.     

Clinical whole exome sequencing revealed de novo heterozygous stop-gain and missense variants in the STXBP1 gene associated with epilepsy in Saudi families

Intellectual disability and developmental encephalopathies are mostly linked with infant epilepsy. Epileptic encephalopathy is a term that is used to define association between developmental delay and epilepsy. Mutations in the STXBP1 (Syntaxin-binding protein 1) gene have been previously reported in association with multiple severe early epileptic encephalopathies along with many neurodevelopmental disorders. Among the disorders produced due to any mutations in the STXBP1 gene is developmental and epileptic encephalopathy 4 (OMIM: 612164), is an autosomal dominant neurologic disorder categorized by the onset of tonic seizures in early infancy (usually in the first months of life). In this article, we report two Saudi families one with de novo heterozygous stop-gain mutation c.364C > T and a novel missense c. 305C > A p.Ala102Glu in exon 5 of the STXBP1 gene (OMIM: 602926) lead to development of epileptic encephalopathy 4. The variants identified in the current study broadened the genetic spectrum of STXBP1 gene related with diseases, which will help to add in the literature and benefit to the studies addressing this disease in the future.